Novel lipids and compositions for the delivery of therapeutics

ABSTRACT

The present invention provides lipids that are advantageously used in lipid particles for the in vivo delivery of therapeutic agents to cells. In particular, the invention provides lipids having the following structures: (Formula (I) or (XXXV)).

GOVERNMENT SUPPORT

The work described herein was carried out, at least in part, using fundsfrom the U.S. Government under grant number HHSN266200600012C awarded bythe National Institute of Allergy and Infectious Diseases. Thegovernment may therefore have certain rights in the invention.

PRIORITY

This application claims priority to U.S. Ser. No. 61/113,179, filed Nov.10, 2008; U.S. Ser. No. 61/154,350, filed Feb. 20, 2009; U.S. Ser. No.61/171,439, filed Apr. 21, 2009; U.S. Ser. No. 61/185,438, filed Jun. 9,2009; U.S. Ser. No. 61/225,898, filed Jul. 15, 2009; and U.S. Ser. No.61/234,098, filed Aug. 14, 2009, the contents of each of which isincorporated herein by reference in its entirety.

BACKGROUND

1. Technical Field

The present invention relates to the field of therapeutic agent deliveryusing lipid particles. In particular, the present invention providescationic lipids and lipid particles comprising these lipids, which areadvantageous for the in vivo delivery of nucleic acids, as well asnucleic acid-lipid particle compositions suitable for in vivotherapeutic use. Additionally, the present invention provides methods ofmaking these compositions, as well as methods of introducing nucleicacids into cells using these compositions, e.g., for the treatment ofvarious disease conditions.

2. Description of the Related Art

Therapeutic nucleic acids include, e.g., small interfering RNA (siRNA),micro RNA (miRNA), antisense oligonucleotides, ribozymes, plasmids,immune stimulating nucleic acids, antisense, antagomir, antimir,microRNA mimic, supermir, U1 adaptor, and aptamer. These nucleic acidsact via a variety of mechanisms. In the case of siRNA or miRNA, thesenucleic acids can down-regulate intracellular levels of specificproteins through a process termed RNA interference (RNAi). Followingintroduction of siRNA or miRNA into the cell cytoplasm, thesedouble-stranded RNA constructs can bind to a protein termed RISC. Thesense strand of the siRNA or miRNA is displaced from the RISC complexproviding a template within RISC that can recognize and bind mRNA with acomplementary sequence to that of the bound siRNA or miRNA. Having boundthe complementary mRNA the RISC complex cleaves the mRNA and releasesthe cleaved strands. RNAi can provide down-regulation of specificproteins by targeting specific destruction of the corresponding mRNAthat encodes for protein synthesis.

The therapeutic applications of RNAi are extremely broad, since siRNAand miRNA constructs can be synthesized with any nucleotide sequencedirected against a target protein. To date, siRNA constructs have shownthe ability to specifically down-regulate target proteins in both invitro and in vivo models. In addition, siRNA constructs are currentlybeing evaluated in clinical studies.

However, two problems currently faced by siRNA or miRNA constructs are,first, their susceptibility to nuclease digestion in plasma and, second,their limited ability to gain access to the intracellular compartmentwhere they can bind RISC when administered systemically as the freesiRNA or miRNA. These double-stranded constructs can be stabilized byincorporation of chemically modified nucleotide linkers within themolecule, for example, phosphothioate groups. However, these chemicalmodifications provide only limited protection from nuclease digestionand may decrease the activity of the construct. Intracellular deliveryof siRNA or miRNA can be facilitated by use of carrier systems such aspolymers, cationic liposomes or by chemical modification of theconstruct, for example by the covalent attachment of cholesterolmolecules. However, improved delivery systems are required to increasethe potency of siRNA and miRNA molecules and reduce or eliminate therequirement for chemical modification.

Antisense oligonucleotides and ribozymes can also inhibit mRNAtranslation into protein. In the case of antisense constructs, thesesingle stranded deoxynucleic acids have a complementary sequence to thatof the target protein mRNA and can bind to the mRNA by Watson-Crick basepairing. This binding either prevents translation of the target mRNAand/or triggers RNase H degradation of the mRNA transcripts.Consequently, antisense oligonucleotides have tremendous potential forspecificity of action (i.e., down-regulation of a specificdisease-related protein). To date, these compounds have shown promise inseveral in vitro and in vivo models, including models of inflammatorydisease, cancer, and HIV (reviewed in Agrawal, Trends in Biotech.14:376-387 (1996)). Antisense can also affect cellular activity byhybridizing specifically with chromosomal DNA. Advanced human clinicalassessments of several antisense drugs are currently underway. Targetsfor these drugs include the bcl2 and apolipoprotein B genes and mRNAproducts.

Immune-stimulating nucleic acids include deoxyribonucleic acids andribonucleic acids. In the case of deoxyribonucleic acids, certainsequences or motifs have been shown to illicit immune stimulation inmammals. These sequences or motifs include the CpG motif,pyrimidine-rich sequences and palindromic sequences. It is believed thatthe CpG motif in deoxyribonucleic acids is specifically recognized by anendosomal receptor, toll-like receptor 9 (TLR-9), which then triggersboth the innate and acquired immune stimulation pathway. Certain immunestimulating ribonucleic acid sequences have also been reported. It isbelieved that these RNA sequences trigger immune activation by bindingto toll-like receptors 6 and 7 (TLR-6 and TLR-7). In addition,double-stranded RNA is also reported to be immune stimulating and isbelieve to activate via binding to TLR-3.

One well known problem with the use of therapeutic nucleic acids relatesto the stability of the phosphodiester internucleotide linkage and thesusceptibility of this linker to nucleases. The presence of exonucleasesand endonucleases in serum results in the rapid digestion of nucleicacids possessing phosphodiester linkers and, hence, therapeutic nucleicacids can have very short half-lives in the presence of serum or withincells. (Zelphati, O., et al., Antisense. Res. Dev. 3:323-338 (1993); andThierry, A. R., et al., pp 147-161 in Gene Regulation: Biology ofAntisense RNA and DNA (Eds. Erickson, R P and Izant, J G; Raven Press,NY (1992)). Therapeutic nucleic acid being currently being developed donot employ the basic phosphodiester chemistry found in natural nucleicacids, because of these and other known problems.

This problem has been partially overcome by chemical modifications thatreduce serum or intracellular degradation. Modifications have beentested at the internucleotide phosphodiester bridge (e.g., usingphosphorothioate, methylphosphonate or phosphoramidate linkages), at thenucleotide base (e.g., 5-propynyl-pyrimidines), or at the sugar (e.g.,2′-modified sugars) (Uhlmann E., et al. Antisense: ChemicalModifications. Encyclopedia of Cancer, Vol. X., pp 64-81 Academic PressInc. (1997)). Others have attempted to improve stability using 2′-5′sugar linkages (see, e.g., U.S. Pat. No. 5,532,130). Other changes havebeen attempted. However, none of these solutions have proven entirelysatisfactory, and in vivo free therapeutic nucleic acids still have onlylimited efficacy.

In addition, as noted above relating to siRNA and miRNA, problems remainwith the limited ability of therapeutic nucleic acids to cross cellularmembranes (see, Vlassov, et al., Biochim. Biophys. Acta 1197:95-1082(1994)) and in the problems associated with systemic toxicity, such ascomplement-mediated anaphylaxis, altered coagulatory properties, andcytopenia (Galbraith, et al., Antisense Nucl. Acid Drug Des. 4:201-206(1994)).

To attempt to improve efficacy, investigators have also employedlipid-based carrier systems to deliver chemically modified or unmodifiedtherapeutic nucleic acids. In Zelphati, O. and Szoka, F. C., J. Contr.Rel. 41:99-119 (1996), the authors refer to the use of anionic(conventional) liposomes, pH sensitive liposomes, immunoliposomes,fusogenic liposomes, and cationic lipid/antisense aggregates. SimilarlysiRNA has been administered systemically in cationic liposomes, andthese nucleic acid-lipid particles have been reported to provideimproved down-regulation of target proteins in mammals includingnon-human primates (Zimmermann et al., Nature 441: 111-114 (2006)).

In spite of this progress, there remains a need in the art for improvedlipid-therapeutic nucleic acid compositions that are suitable forgeneral therapeutic use. Preferably, these compositions wouldencapsulate nucleic acids with high-efficiency, have high drug:lipidratios, protect the encapsulated nucleic acid from degradation andclearance in serum, be suitable for systemic delivery, and provideintracellular delivery of the encapsulated nucleic acid. In addition,these lipid-nucleic acid particles should be well-tolerated and providean adequate therapeutic index, such that patient treatment at aneffective dose of the nucleic acid is not associated with significanttoxicity and/or risk to the patient. The present invention provides suchcompositions, methods of making the compositions, and methods of usingthe compositions to introduce nucleic acids into cells, including forthe treatment of diseases.

BRIEF SUMMARY

The present invention provides novel cationic lipids, as well as lipidparticles comprising the same. These lipid particles may furthercomprise an active agent and be used according to related methods of theinvention to deliver the active agent to a cell.

In one aspect, the invention provides lipids having the structure offormula I:

salts or isomers thereof, wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl,or -linker-ligand;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocyclealkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle, or linker-ligand;

X and Y are each independently —O—, —S—, alkylene, —N(Q)-, —C(O)—,—O(CO), —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—, —OS(O)(Q₂)O—, or—OP(O)(Q₂)O—;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl;

Q₁ is independently for each occurrence O or S;

Q₂ is independently for each occurrence O, S, N(Q)(Q), alkyl or alkoxy;

A₁, and A₂ are each independently —O—, —S—, —CH₂—, —CHR⁵—, —CR⁵R⁵—,—CHF— or —CF₂—;

Z is N, or C(R₃); and

m and n are each independently 0 to 5, where m and n taken togetherresult in a 3, 4, 5, 6, 7 or 8 member ring.

In another aspect, the invention provides a lipid having the structureof formula XXXV:

salts or isomers thereof, wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl,or -linker-ligand;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocyclealkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle, or linker-ligand; or,

each R₃ taken together with the atom to which they are attached are a3-8 membered optionally substituted cycloalkyl group or a 3-8 memberedoptionally substituted heterocycle group;

X and Y are each independently —O—, —S—, alkylene, —N(Q)-, —C(O)—,—O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—, —OS(O)(Q₂)O—, or—OP(O)(Q₂)O—;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl;

Q₁ is independently for each occurrence O or S; and,

Q₂ is independently for each occurrence O, S, N(Q)Q), alkyl or alkoxy;

In another aspect, the invention provides a lipid particle comprisingthe lipids of the present invention. In certain embodiments, the lipidparticle further comprises a neutral lipid and a lipid capable ofreducing particle aggregation. In one embodiment, the lipid particleconsists essentially of (i) at least one lipid of the present invention;(ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii)sterol, e.g. cholesterol; and (iv) peg-lipid, e.g. PEG-DMG or PEG-DMA,in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid:25-55% sterol; 0.5-15% PEG-lipid. In one embodiment, the lipid of thepresent invention is optically pure.

In one embodiment, the lipid particle comprises at least two lipidsdisclosed herein. For example, a mixture of cationic lipids can be usedin a lipid particle, such that the mixture comprises 20-60% of the totallipid content on a molar basis.

In additional related embodiments, the present invention includes lipidparticles of the invention that further comprise therapeutic agent. Inone embodiment, the therapeutic agent is a nucleic acid. In oneembodiment, the nucleic acid is a plasmid, an immunostimulatoryoligonucleotide, a single stranded oligonucleotide, e.g. an antisenseoligonucleotide, an antagomir; a double stranded oligonucleotide, e.g. asiRNA; an aptamer or a ribozyme.

In yet another related embodiment, the present invention includes apharmaceutical composition comprising a lipid particle of the presentinvention and a pharmaceutically acceptable excipient, carrier ofdiluent.

The present invention further includes, in other related embodiments, amethod of modulating the expression of a target gene in a cell, themethod comprising providing to a cell a lipid particle or pharmaceuticalcomposition of the present invention. The target gene can be a wild typegene. In another embodiment, the target gene contains one or moremutations. In a particular embodiment, the method comprises specificallymodulating expression of a target gene containing one or more mutations.In particular embodiments, the lipid particle comprises a therapeuticagent selected from an immunostimulatory oligonucleotide, a singlestranded oligonucleotide, e.g. an antisense oligonucleotide, anantagomir; a double stranded oligonucleotide, e.g. a siRNA, an aptamer,a ribozyme. In one embodiment, the nucleic acid is plasmid that encodesa siRNA, an antisense oligonucleotide, an aptamer or a ribozyme.

In one aspect of the invention, the target gene is selected from thegroup consisting of Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV,PDGF beta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene,MEKK gene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene,JUN gene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene,Cyclin A gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene,PKC gene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, SORT1gene, XBP1 gene, topoisomerase I gene, topoisomerase II alpha gene, p73gene, p21(WAF1/CIP1) gene, p27(KIP1) gene, PPM1D gene, RAS gene,caveolin I gene, MIB I gene, MTAI gene, M68 gene, mutations in tumorsuppressor genes, p53 tumor suppressor gene, and combinations thereof.

In another embodiment, the nucleic acid is a plasmid that encodes apolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In yet a further related embodiment, the present invention includes amethod of treating a disease or disorder characterized by overexpressionof a polypeptide in a subject, comprising providing to the subject alipid particle or pharmaceutical composition of the present invention,wherein the therapeutic agent is selected from an siRNA, a microRNA, anantisense oligonucleotide, and a plasmid capable of expressing an siRNA,a microRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In a further embodiment, the present invention includes a method ofinducing an immune response in a subject, comprising providing to thesubject a pharmaceutical composition of the present invention, whereinthe therapeutic agent is an immunostimulatory oligonucleotide. Inparticular embodiments, the pharmaceutical composition is provided tothe patient in combination with a vaccine or antigen.

In a related embodiment, the present invention includes a vaccinecomprising the lipid particle of the present invention and an antigenassociated with a disease or pathogen. In one embodiment, the lipidparticle comprises an immunostimulatory nucleic acid or oligonucleotide.In a particular embodiment, the antigen is a tumor antigen. In anotherembodiment, the antigen is a viral antigen, a bacterial antigen, or aparasitic antigen.

The present invention further includes methods of preparing the lipidparticles and pharmaceutical compositions of the present invention, aswell as kits useful in the preparation of these lipid particle andpharmaceutical compositions.

In another aspect, the invention provides a method of evaluating acomposition that includes an agent, e.g. a therapeutic agent ordiagnostic agent, and a lipid of the present invention.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1. Schematic representation of an optically pure lipid withconjugated targeting ligands.

FIG. 2. Schematic representation of an optically pure lipid withconjugated targeting ligands.

FIG. 3. Schematic representation of racemic lipids with conjugatedtargeting ligands.

FIG. 4. Shows the results of in vivo modulation of FVII gene usingformulations comprising the lipids 506, 512 or 519.

FIG. 5. Schematic representation of features of the lipids of thepresent invention.

FIG. 6 shows a graph illustrating the relative FVII protein levels inanimals administered with 0.05 or 0.005 mg/kg of lipid particlescontaining different cationic lipids.

DETAILED DESCRIPTION

The present invention is based, in part, upon the discovery of cationiclipids that provide advantages when used in lipid particles for the invivo delivery of a therapeutic agent. In particular, as illustrated bythe accompanying Examples, the present invention provides nucleicacid-lipid particle compositions comprising a cationic lipid accordingto the present invention. In some embodiments, a composition describedherein provides increased activity of the nucleic acid and/or improvedtolerability of the compositions in vivo, which can result in asignificant increase in therapeutic index as compared to lipid-nucleicacid particle compositions previously described. Additionallycompositions and methods of use are disclosed that can provide foramelioration of the toxicity observed with certain therapeutic nucleicacid-lipid particles.

In certain embodiments, the present invention specifically provides forimproved compositions for the delivery of siRNA molecules. It is shownherein that these compositions are effective in down-regulating theprotein levels and/or mRNA levels of target proteins. Furthermore, it isshown that the activity of these improved compositions is dependent onthe presence of a certain cationic lipids and that the molar ratio ofcationic lipid in the formulation can influence activity.

The lipid particles and compositions of the present invention may beused for a variety of purposes, including the delivery of associated orencapsulated therapeutic agents to cells, both in vitro and in vivo.Accordingly, the present invention provides methods of treating diseasesor disorders in a subject in need thereof, by contacting the subjectwith a lipid particle of the present invention associated with asuitable therapeutic agent.

As described herein, the lipid particles of the present invention areparticularly useful for the delivery of nucleic acids, including, e.g.,siRNA molecules and plasmids. Therefore, the lipid particles andcompositions of the present invention may be used to modulate theexpression of target genes and proteins both in vitro and in vivo bycontacting cells with a lipid particle of the present inventionassociated with a nucleic acid that reduces target gene expression(e.g., an siRNA) or a nucleic acid that may be used to increaseexpression of a desired protein (e.g., a plasmid encoding the desiredprotein).

Various exemplary embodiments of the cationic lipids of the presentinvention, as well as lipid particles and compositions comprising thesame, and their use to deliver therapeutic agents and modulate gene andprotein expression are described in further detail below.

Lipids

The present invention provides novel lipids having certain designfeatures. As shown in FIG. 5, the lipid design features include at leastone of the following: a head group with varying pKa, a cationic, 1°, 2°and 3°, monoamine, Di and triamine, Oligoamine/polyamine, a low pKa headgroups—imidazoles and pyridine, guanidinium, anionic, zwitterionic andhydrophobic tails can include symmetric and/or unsymmetric chains, longand shorter, saturated and unsaturated chain the back bone includesBackbone glyceride and other acyclic analogs, cyclic, spiro, bicyclicand polycyclic linkages with ethers, esters, phosphate and analogs,sulfonate and analogs, disulfides, pH sensitive linkages like acetalsand ketals, imines and hydrazones, and oximes.

The present invention provides novel lipids that are advantageously usedin lipid particles of the present invention for the in vivo delivery oftherapeutic agents to cells, including lipids having the followingstructure

salts or isomers thereof wherein:

-   -   cy is optionally substituted cyclic, optionally substituted        heterocyclic or heterocycle, optionally substituted aryl or        optionally substituted heteroaryl;    -   R₁ and R₂ are each independently for each occurrence optionally        substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀        alkenyl, optionally substituted C₁₀-C₃₀ alkynyl, optionally        substituted C₁₀-C₃₀ acyl or -linker-ligand;    -   X and Y are each independently O or S, alkyl or N(Q); and    -   Q is H, alkyl, acyl, ω-aminoalkyl, ω-(substituted)aminoalky,        ω-phosphoalkyl or ω-thiophosphoalkyl.

In one embodiment, the lipid has the structure

salts or isomers thereof, wherein:

-   -   -   R₁ and R₂ are each independently for each occurrence            optionally substituted C₁₀-C₃₀ alkyl, optionally substituted            C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkynyl,            optionally substituted C₁₀-C₃₀ acyl or -linker-ligand;

    -   X and Y are each independently O or S, alkyl or N(Q);

    -   Q is H, alkyl, acyl, alkylamino or alkylphosphate; and

    -   R^(A) and R^(B) are each independently H, R₃, —Z′—R₃,        -(A₂)_(j)—Z′—R₃, acyl, sulfonate or

-   -   Q₁ is independently for each occurrence O or S;    -   Q₂ is independently for each occurrence O, S, N(Q), alkyl or        alkoxy;    -   Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky,        ω-phosphoalkyl or ω-thiophosphoalkyl;    -   A₁, A₄, and A₅ are each independently O, S, CH₂, CHF or CF₂;    -   Z′ is O, S, N(Q) or alkyl;    -   i and j are independently 0 to 10; and    -   R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally        substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀        alkenyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,        alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,        ω-(substituted)aminoalkyls, ω-phosphoalkyls,        ω-thiophosphoalkyls, polyethylene glycol (PEG, mw 100-40K), mPEG        (mw 120-40K), heteroaryl, heterocycle or linker-ligand.

In another aspect, the lipid has one of the following structures, saltsor isomers thereof:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl, or -linker-ligand;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls,ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls,ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl,heterocycle, or linker-ligand;

R⁴ is independently for each occurrence H, ═O, OR₃ or R₃;

X and Y are each independently O, S, alkyl or N(Q);

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky, ω-phosphoalkyl orω-thiophosphoalkyl;

Q₁ is independently for each occurrence O or S;

Q₂ is independently for each occurrence O, S, N(Q), alkyl or alkoxy;

A₁, A₂, A₃, A₄, A₅ and A₆ are each independently O, S, CH₂, CHF or CF₂;

A₇ is O, S or N(Q);

A₈ is independently for each occurrence CH₂, CHF or CF₂;

A₉ is —C(O)— or —C(H)(R₃)—;

E and F are each independently for each occurrence O, S, N(Q), C(O),C(O)O, C(O)N, S(O), S(O)₂, SS, O═N, aryl, heteroaryl, cyclic orheterocycle

Z is N, C(R₃);

Z′ is O, S, N(Q) or alkyl;

k is 0, 1 or 2;

m and n are 0 to 5, where m and n taken together result in a 3, 4, 5, 6,7 or 8 member ring;

p is 1-5;

q is 0-5, where p and q taken together result in a 3, 4, 5, 6, 7 or 8member ring

i and j are 0-10; and

a and b are 0-2.

In one embodiment, X and Y can be independently (CO), O(CO), O(CO)N,N(CO)O, (CO)O, O(CO)O, a sulfonate, or a phosphate.

In one embodiment, R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl,or -linker-ligand.

In one embodiment, R₃ is independently for each occurrence H, optionallysubstituted C₁-C₁₀ alkyl, optionally substituted C₂-C₁₀ alkenyl,optionally substituted C₂-C₁₀ alkynyl, optionally substitutedalkylheterocycle, optionally substituted heterocyclealkyl, optionallysubstituted alkylphosphate, optionally substituted phosphoalkyl,optionally substituted alkylphosphorothioate, optionally substitutedphosphorothioalkyl, optionally substituted alkylphosphorodithioate,optionally substituted phosphorodithioalkyl, optionally substitutedalkylphosphonate, optionally substituted phosphonoalkyl, optionallysubstituted amino, optionally substituted alkylamino, optionallysubstituted di(alkyl)amino, optionally substituted aminoalkyl,optionally substituted alkylaminoalkyl, optionally substituteddi(alkyl)aminoalkyl, optionally substituted hydroxyalkyl, optionallysubstituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), optionally substituted heteroaryl, oroptionally substituted heterocycle, or linker-ligand.

In one embodiment, X and Y are each independently —O—, —S—, alkylene,—N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O, —OC(O)O—,—OS(O)(Q₂)O—, or —OP(O)(Q₂)O—.

In one embodiment, Q is H, alkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl.

In one embodiment, Q₂ is independently for each occurrence O, S, N(Q)Q),alkyl or alkoxy,

In one embodiment, A₁, A₂, A₃, A₄, A₅ and A₆ are each independently —O—,—S—, —CH₂—, —CHR⁵—, —CR⁵R⁵—, —CHF— or —CF₂—.

In one embodiment, A₈ is independently for each occurrence —CH₂—,—CHR⁵—, —CR⁵R⁵—, —CHF—, or —CF₂—.

In one embodiment, E and F are each independently for each occurrence—O—, —S—, —N(Q)-, —C(O)—, —C(O)O—, —OC(O)—, —C(O)N(Q)-, —N(Q)C(O)—,—S(O)—, —S(O)₂—, —SS—, —O—N═, ═N—O—, arylene, heteroarylene,cycloalkylene, or heterocyclylene.

In one embodiment, Z is N, or C(R₃).

In one embodiment, Z′ is —O—, —S—, —N(Q)-, or alkylene.

In one embodiment, R⁵ is H, halo, cyano, hydroxy, amino, optionallysubstituted alkyl, optionally substituted alkoxy, or optionallysubstituted cycloalkyl.

In one embodiment, i and j are each independently 0-10.

In one embodiment, a and b are each independently 0-2.

In some circumstances, R₃ is ω-aminoalkyl, ω-(substituted)aminoalkyl,ω-phosphoalkyl, or ω-thiophosphoalkyl; each of which is optionallysubstituted. Examples of ω-(substituted)aminoalkyl groups include2-(dimethylamino)ethyl, 3-(diisopropylamino)propyl, or3-(N-ethyl-N-isopropylamino)-1-methylpropyl.

In one embodiment, X and Y can be independently —O—, —S—, alkylene, or—N(Q)-.

It has been found that cationic lipids comprising unsaturated alkylchains are particularly useful for forming lipid nucleic acid particleswith increased membrane fluidity. In one embodiment, at least one of R₁or R₂ comprises at least one, at least two or at least three sites ofunsaturation, e.g. double bond or triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one, at leasttwo or at least three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise at least one, at least two orat least three sites of unsaturation.

In one embodiment, R₁ and R₂ comprise different numbers of unsaturation,e.g., one of R₁ and R₂ has one site of unsaturation and the other hastwo or three sites of unsaturation.

In one embodiment, R₁ and R₂ both comprise the same number ofunsaturation sites.

In one embodiment, R₁ and R₂ comprise different types of unsaturation,e.g. unsaturation in one of R₁ and R₂ is double bond and in the otherunsaturation is triple bond.

In one embodiment, R₁ and R₂ both comprise the same type ofunsaturation, e.g. double bond or triple bond.

In one embodiment, at least one of R₁ or R₂ comprises at least onedouble bond and at least one triple bond.

In one embodiment, only one of R₁ or R₂ comprises at least one doublebond and at least one triple bond.

In one embodiment, R₁ and R₂ both comprise at least one double bond andat least one triple bond.

In one embodiment, R₁ and R₂ are both same, e.g. R₁ and R₂ are bothlinoleyl (C18) or R₁ and R₂ are both heptadeca-9-enyl.

In one embodiment, R₁ and R₂ are different from each other.

In one embodiment, at least one of R₁ and R₂ is cholesterol.

In one embodiment, one of R₁ and R₂ is -linker-ligand.

In one embodiment, one of R₁ and R₂ is -linker-ligand and ligand is alipophile.

In one embodiment, at least one of R₁ or R₂ comprises at least one CH₂group with one or both H replaced by F, e.g. CHF or CF₂. In oneembodiment, both R₁ and R₂ comprise at least one CH₂ group with one ortwo H replaced by F, e.g. CHF or CF₂.

In one embodiment, only one of R₁ and R₂ comprises at least one CH₂group with one or both H replaced by F.

In one embodiment, at least one of R₁ or R₂ terminates in CH₂F, CHF₂ orCF₃. In one embodiment, both R₁ and R₂ terminate in CH₂F, CHF₂ or CF₃.

In one embodiment, at least one of R₁ or R₂ is—(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—Z″—(CH₂)_(y)—CH₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is—(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃, wherein each y is independently 1-10 and Z″is O, S or N(Q).

In one embodiment, both of R₁ and R₂ are —(CH₂)_(y)—Z″—(CF₂)_(y)—CF₃,wherein each y is independently 1-10 and Z″ is O, S or N(Q).

In one embodiment, at least one of R₁ or R₂ is —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In one embodiment, both of R₁ and R₂ are —(CF₂)_(y)—(CF₂)_(y)—CF₃,wherein each y is independently 1-10.

In one embodiment,

is selected from the group consisting of:

In one embodiment, R₃ is chosen from a group consisting of methyl,ethyl, polyamine, —(CH₂)_(h)-heteroaryl, —(CH₂)_(h)—N(Q)₂, —O—N(Q)₂,—(CH₂)_(h)—Z′—(CH₂)_(h)-heteroaryl, linker-ligand,—(CH₂)_(h)-heterocycle, and —(CH₂)_(h)—Z″—(CH₂)_(h)-heterocycle, whereineach h is independently 0-13 and Z″ is O, S or N(Q).

In one embodiment, when Z is C(R₃), at least one R₃ is co-aminoalkyl orω-(substituted)aminoalkyl.

In one embodiment, when Z′ is O, S or alkyl, at least one R₃ isω-aminoalkyl or ω-(substituted)aminoalkyl.

In one embodiment, Q is linker-ligand.

In one embodiment, ligand is fusogenic peptide.

In one embodiment, the lipid is a racemic mixture.

In one embodiment, the lipid is enriched in one diastereomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%diastereomeric excess.

In one embodiment, the lipid is enriched in one enantiomer, e.g. thelipid has at least 95%, at least 90%, at least 80% or at least 70%enantiomer excess.

In one embodiment, the lipid is chirally pure, e.g. is a single opticalisomer.

In one embodiment, the lipid is enriched for one optical isomer.

Where a double bond is present (e.g., a carbon-carbon double bond orcarbon-nitrogen double bond), there can be isomerism in theconfiguration about the double bond (i.e. cis/trans or E/Z isomerism).Where the configuration of a double bond is illustrated in a chemicalstructure, it is understood that the corresponding isomer can also bepresent. The amount of isomer present can vary, depending on therelative stabilities of the isomers and the energy required to convertbetween the isomers. Accordingly, some double bonds are, for practicalpurposes, present in only a single configuration, whereas others (e.g.,where the relative stabilities are similar and the energy of conversionlow) may be present as inseparable equilibrium mixture ofconfigurations.

In another aspect, the invention features a compound of formula XXXIVa,XXXIVb, XXXIVc, XXXIVd, or XXXIVe, salts or isomers thereof:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl, oroptionally substituted C₁₀-C₃₀ alkynyl;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocyclealkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle; and

n is 1, 2, or 3.

In some embodiments, R₃ is optionally substituted heterocyclealkyl,optionally substituted amino, optionally substituted alkylamino,optionally substituted di(alkyl)amino, optionally substitutedaminoalkyl, optionally substituted alkylaminoalkyl, optionallysubstituted di(alkyl)aminoalkyl, or optionally substituted heterocycle.

In one aspect, the lipid is a compound of formula XIIIa:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl, oroptionally substituted C₁₀-C₃₀ alkynyl;

R₃ and R_(3′) are independently for each occurrence H, optionallysubstituted C₁-C₁₀ alkyl, optionally substituted C₂-C₁₀ alkenyl,optionally substituted C₂-C₁₀ alkynyl, optionally substitutedalkylheterocycle, optionally substituted heterocyclealkyl, optionallysubstituted alkylphosphate, optionally substituted phosphoalkyl,optionally substituted alkylphosphorothioate, optionally substitutedphosphorothioalkyl, optionally substituted alkylphosphorodithioate,optionally substituted phosphorodithioalkyl, optionally substitutedalkylphosphonate, optionally substituted phosphonoalkyl, optionallysubstituted amino, optionally substituted alkylamino, optionallysubstituted di(alkyl)amino, optionally substituted aminoalkyl,optionally substituted alkylaminoalkyl, optionally substituteddi(alkyl)aminoalkyl, optionally substituted hydroxyalkyl, optionallysubstituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), optionally substituted heteroaryl, oroptionally substituted heterocycle;

or R₃ and R_(3′) can be taken together with the atoms to which they areattached to form an optionally substituted carbocyclyl, optionallysubstituted heterocyclyl, optionally substituted aryl or optionallysubstituted heteroaryl; each of which is substituted with 0-4occurrences of R₄;

each R₄ is independently selected from optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted amino, optionally substitutedalkylamino, optionally substituted di(alkyl)amino, optionallysubstituted aminoalkyl, optionally substituted alkylaminoalkyl,optionally substituted di(alkyl)aminoalkyl, optionally substitutedhydroxyalkyl, optionally substituted aryl, optionally substitutedheteroaryl, or optionally substituted heterocycle;

X and Y are each independently —O—, —S—, alkylene, or —N(Q)-;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalkyl, ω-phosphoalkyl,or ω-thiophosphoalkyl;

A₁ and A₂ are each independently —O—, —S—, or —CR⁵R⁵—; and

R⁵ is H, halo, cyano, hydroxy, amino, optionally substituted alkyl,optionally substituted alkoxy, or optionally substituted cycloalkyl; and

Z and Z′ are each independently selected from —O—, —S—, —N(Q)-, alkyleneor absent; and

a and b are each independently 0-2.

In some embodiments, X and Y are each independently O.

In some embodiments, the sum of a and b is 1, 2, or 3.

In some embodiments, A₁ and A₂ are each independently —CR⁵R⁵—.

In some embodiments, Z and Z′ are each a bond.

In some embodiments, R₃ and R_(3′) can be taken together with the atomsto which they are attached to form an optionally substitutedcarbocyclyl, optionally substituted heterocyclyl, optionally substitutedaryl or optionally substituted heteroaryl.

In some embodiments, R₃ and R_(3′) can be taken together with the atomsto which they are attached to form an optionally substituted carbocyclyl(e.g., optionally substituted with amino, alkylamino, or dialkylamino).

In some embodiments, R₃ and R_(3′) can be taken together with the atomsto which they are attached to form an optionally substitutedheterocyclyl (e.g., a nitrogen containing heterocyclyl).

In some embodiments, R₃ and R_(3′) are taken together to form acarbocyclic ring (e.g., cyclohexyl) substituted with 0-3 occurrence ofR₄.

In some embodiments, R₃ and R_(3′) are taken together to form aheterocyclic ring (e.g., piperidine) substituted with 0-3 occurrences ofR₄.

In some embodiments, each R₄ is independently selected from optionallyoptionally substituted amino, optionally substituted alkylamino,optionally substituted di(alkyl)amino, optionally substitutedaminoalkyl, optionally substituted alkylaminoalkyl, optionallysubstituted di(alkyl)aminoalkyl, and optionally substitutedhydroxyalkyl.

In one aspect, the lipid is a compound of formula XXXIX, salts orisomers thereof:

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl, or -linker-ligand;

R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls,ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls,ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl,heterocycle, or linker-ligand;

X and Y are each independently O, C(O)O, S, alkyl or N(Q);

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky, ω-phosphoalkyl orω-thiophosphoalkyl;

In one aspect, the lipid is a compound of formula XXXIII, salts orisomers thereof

wherein:

R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkenyl,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀acyl, or -linker-ligand;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), heteroaryl, heterocycle, orlinker-ligand;

E is O, S, N(Q), C(O)O, C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q),S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O═N, aryl, heteroaryl, cyclic orheterocycle; and,

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky, ω-phosphoalkyl orω-thiophosphoalkyl.

In one embodiment, R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl.

In another embodiment, R₃ is H, optionally substituted C₁-C₁₀ alkyl,optionally substituted C₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀alkynyl, optionally substituted alkylheterocycle, optionally substitutedheterocyclealkyl, optionally substituted alkylphosphate, optionallysubstituted phosphoalkyl, optionally substituted alkylphosphorothioate,optionally substituted phosphorothioalkyl, optionally substitutedalkylphosphorodithioate, optionally substituted phosphorodithioalkyl,optionally substituted alkylphosphonate, optionally substitutedphosphonoalkyl, optionally substituted amino, optionally substitutedalkylamino, optionally substituted di(alkyl)amino, optionallysubstituted aminoalkyl, optionally substituted alkylaminoalkyl,optionally substituted di(alkyl)aminoalkyl, optionally substitutedhydroxyalkyl, optionally substituted polyethylene glycol (PEG, mw100-40K), optionally substituted mPEG (mw 120-40K), optionallysubstituted heteroaryl, optionally substituted heterocycle, orlinker-ligand.

In yet another embodiment, E is —O—, —S—, —N(Q)-, —C(O)O—, —OC(O)—,—C(O)—, —N(Q)C(O)—, —C(O)N(Q)-, —N(Q)C(O)O—, —OC(O)N(Q)-, S(O),—N(Q)S(O)₂N(Q)-, —S(O)₂—, —N(Q)S(O)₂—, —SS—, —O—N═, ═N—O—,—C(O)—N(Q)-N═, —N(Q)-N═, —N(Q)-O—, —C(O)S—, arylene, heteroarylene,cyclalkylene, or heterocyclylene.

In another embodiment, Q is H, alkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl or ω-thiophosphoalkyl.

In one embodiment, where the lipid is a compound of formula XXXIII,provided that when E is C(O)O and R³ is

R¹ and R² are not both linoleyl.

In another embodiment, the lipid is a compound of formula XXXIII,wherein E is O, S, N(Q), C(O), N(Q)C(O), C(O)N(Q), (O)N(CO)O, O(CO)N(Q),S(O), NS(O)2N(Q), S(O)₂, N(Q)S(O)2, SS, O═N, aryl, heteroaryl, cyclic orheterocycle.

In one embodiment, the lipid is a compound of formula XXXIII, wherein R₃is H, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, alkylhetrocycle, alkylphosphate, alkylphosphorothioate,alkylphosphorodithioate, alkylphosphonates, alkylamines, hydroxyalkyls,ω-aminoalkyls, ω-(substituted)aminoalkyls, ω-phosphoalkyls,ω-thiophosphoalkyls, optionally substituted polyethylene glycol (PEG, mw100-40K), optionally substituted mPEG (mw 120-40K), heteroaryl,heterocycle, or linker-ligand.

In yet another embodiment, the lipid is a compound of formula XXXIII,wherein R₁ and R₂ are each independently for each occurrence optionallysubstituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkynyl,optionally substituted C₁₀-C₃₀ acyl, or -linker-ligand.

In one embodiment, the invention features a lipid of formula XXXVIII:

wherein

E is O, S, N(Q), C(O)O, C(O), N(Q)C(O), C(O)N(Q), (Q)N(CO)O, O(CO)N(Q),S(O), NS(O)2N(Q), S(O)2, N(Q)S(O)2, SS, O═N, aryl, heteroaryl, cyclic orheterocycle;

Q is H, alkyl, ω-aminoalkyl, ω-(substituted)aminoalky, ω-phosphoalkyl orω-thiophosphoalkyl;

R₁ and R₂ and R_(x) are each independently for each occurrence H,optionally substituted C₁-C₁₀ alkyl, optionally substituted C₁₀-C₃₀alkyl, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀ acyl, or linker-ligand,provided that at least one of R₁, R₂ and R_(x) is not H;

R₃ is H, optionally substituted C₁-C₁₀ alkyl, optionally substitutedC₂-C₁₀ alkenyl, optionally substituted C₂-C₁₀ alkynyl, alkylhetrocycle,alkylphosphate, alkylphosphorothioate, alkylphosphorodithioate,alkylphosphonates, alkylamines, hydroxyalkyls, ω-aminoalkyls,ω-(substituted)aminoalkyls, ω-phosphoalkyls, ω-thiophosphoalkyls,optionally substituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), heteroaryl, heterocycle, orlinker-ligand;

n is 0, 1, 2, or 3.

In one embodiment, where the lipid is a compound of formula XXXVIII,provided that when E is C(O)O, R³ is

and one of R₁, R₂, or R_(x) is H, then the remaining of R₁, R₂, or R_(x)are not both linoleyl.

In some embodiments, each of R₁ and R₂ is independently for eachoccurrence optionally substituted C₁₀-C₃₀ alkyl, optionally substitutedC₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ acyl, or linker-ligand.

In some embodiments, R_(x) is H or optionally substituted C₁-C₁₀ alkyl.

In some embodiments, R_(x) is optionally substituted C₁₀-C₃₀ alkyl,optionally substituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀alkynyl, optionally substituted C₁₀-C₃₀ acyl, or linker-ligand.

In one aspect, the invention features a lipid of the following formulaXL,

wherein:

Q₁ is O, S, CH₂, CHMe, CMe₂, N(R); Q₂ is O, S, CH₂, CHMe, CMe₂, N(R),C(H)═N—N(R)—, N(R)—N═C(H), —C(H)═N—O—, —O—N═C(H), C(H)═N—N(R)—C(O)—,—C(O)—N(R)—N═C(H) Q₃ and or Q₄ is O, S, N(R), Q₁-C(═Z)Q₂, C(H)═N—N(R)—,N(R)—N═C(H); —C(H)═N—O—, —O—N═C(H); C(H)═N—N(R)—C(O)—,—C(O)—N(R)—N═C(H);

Z=O, S, N(R) or absent and when Z is absent C(═Z) is C(R_(n))₂p is 0 to 20; q is 0 to 10; r is 0 to 6; s is 0 to 6.R′ and/or R′ are: alkyl, substituted alkyls, alkenyls, substitutedalkenyls, alkynyls, substituted alkynyls and combinations thereof withnumber of carbon atoms in the chain varying from 4 to 30. R′ and/or R″with alkenyl chain has at least one C═C or substituted C═C moiety andwhen there is more than one C═C moiety is present they are separated byat least one methylene or substituted methylene group. R′ and/or R″ withalkynyl chain has at least one C≡C moiety and when there is more thanone C≡C moiety is present they are separated by at least one methyleneor substituted methylene group. One or more of methylene or substitutedmethylene is interrupted by hetero atoms such as O, S or N(R). Thedouble bond or bonds in the alkyl chain are all with cis- ortrans-configuration or combination of both. The stereochemistry ofchiral center of formula XL is R, S or racemic.R is H, R′, ω-substituted amino-alkyls, ω-substituted amino-alkenyls,ω-substituted amino-alkynyls with number of carbon atoms in the chainvarying from 1 to 30R₁ to R_(n) each occurrence is R;X is: R, C(O)—NH(R), C(O)NR₂, C(═NR)NH(R), C(═NR)NR₂, N(R)—C(O)Y and Yis independently X.

In one aspect, the invention features a lipid of the following formulaXLI,

wherein:

Q₁ is O, S, CH₂, CHMe, CMe₂, N(R); Q₂ is O, S, CH₂, CHMe, CMe₂, N(R),C(H)═N—N(R)—, N(R)—N═C(H), —C(H)═N—O—, —O—N═C(H), C(H)═N—N(R)—C(O)—,—C(O)—N(R)—N═C(H)

Q₃ and or Q₄ is O, S, N(R), CH₂, substituted methylene;Q₅ and or Q₆ is O, S, N(R), CH₂, substituted methyleneZ=O, S, N(R) or absent and when Z is absent C(═Z) is C(R_(n))₂p is 0 to 20; q is 0 to 10; r is 0 to 6; s is 0 to 6.R′ and/or R′ are: alkyl, substituted alkyls, alkenyls, substitutedalkenyls, alkynyls, substituted alkynyls and combinations thereof withnumber of carbon atoms in the chain varying from 4 to 30. R′ and/or R″with alkenyl chain has at least one C═C or substituted C═C moiety andwhen there is more than one C═C moiety is present they are separated byat least one methylene or substituted methylene group. R′ and/or R″ withalkynyl chain has at least one C≡C moiety and when there is more thanone C≡C moiety is present they are separated by at least one methyleneor substituted methylene group. One or more of methylene or substitutedmethylene is interrupted by hetero atoms such as O, S or N(R). Thedouble bond or bonds in the alkyl chain are all with cis- ortrans-configuration or combination of both. The stereochemistry ofchiral center of formula XLI is R, S or racemic.R is H, R′, ω-substituted amino-alkyls, ω-substituted amino-alkenyls,ω-substituted amino-alkynyls with number of carbon atoms in the chainvarying from 1 to 30 R₁ to R_(n) each occurrence is R;X is: R, C(O)—NH(R), C(O)NR₂, C(═NR)NH(R), C(═NR)NR₂, N(R)—C(O)Y and Yis independently X.

In another aspect, the invention features a lipid of one of thefollowing formula XLII, XLIII, XLIV, XLV, XLVI, XLVII, XLVIII, or XLIX

Wherein the variables recited above are as described herein and whereinfor the compounds of formulae above: p is 0 to 20; q is 0 to 10; r is 0to 6; s is 0 to 6, t is 0 to 6 and u is 0 to 10 and the other variablesare as described above.

The present invention comprises of synthesis of lipids described hereinin racemic as well as in optically pure form.

In one aspect, the invention features a lipid of the formula providedbelow:

X=O, S, CH₂, N(Q₃) where Q is H, Me, Et, —(CH₂)_(r)—N(Q₃, Q₄), Y=O, S,CH₂, N(Q₃) where Q is H, Me, Et, —(CH₂)_(r)—N(Q₃, Q₄); Z=N, CH, C(Me),C(Et); Q₁=O or S; Q₂=O or S; A₁, A₂, ═CH₂, CHF, CF₂; m, n, p and/or q isindependently 0 to 5.

The present invention comprises of synthesis of cationic lipids ofdescribed above in racemic as well as in optically pure form.

R₁, R₂ and R₄ are each independently selected from the group consistingof alkyl groups having about 10 to 30 carbon atoms, wherein R₁, R₂ andR₄ independently comprises of: fully saturated alkyl chain, at least onedouble bond, at least one triple bond, at least one hetero atom, atleast one CF₂, at least one CHF or at least one perfluoroalkylatedchain. CF₂/CHF could be on the lipid anchor or on the core. R₃ isindependently selected from the group consisting of: H and C₁-C₁₀alkyls, C₁-C₁₀ alkenyls, C₁-C₁₀ alkynyls, alkylheterocycles,alkylphospates, alkylphosphorothioate, alkylphosphonates, alkylamines,hydroxyalkyls, ω-aminoalkyls, ω-(substituted)aminoalkyls,ω-phosphoalkyls, ω-thiophosphoalkyls, PEG with MW range from 100-40000,mPEG with MW range from 120-40000, heterocycles such as imidazoles,triazoles, pyridines, pyrimidines, purines, substituted pyridines,alkyl/PEG spacer containing receptor targeting ligands such as GalNAc,folic acid, mannose, Fucose, naproxen, ibuprofen and the ligands includesmall molecules that binds to chemokines, integrins, somatostatin,androgen and CNS receptors. R₃ also covers above ligands without spacerbetween the lipids core/anchor.

In one embodiment, the cationic lipid is chosen from a group consistingof lipids shown in Table 1 below.

TABLE 1 Some cationic lipids of the present invention.

Although not all diasteromers for a lipid are shown, one aspect of thepresent invention is to provide all diastereomers and as such chirallypure and diastereomerically enriched lipids are also part of thisinvention.

In one embodiment, R₃ is -linker-ligand.

In one embodiment, the lipid is a targeting cationic lipid and chosenfrom a group consisting of lipids shown in Table 2 below.

TABLE 2 Targeting lipids. # Structure 1.

2.

3.

4.

5.

6.

Although not all diasteromers for a lipid are shown, one aspect of thepresent invention is to provide all diastereomers and as such chirallypure and diastereomerically enriched lipids are also part of thisinvention.

In particular embodiments, the lipids of the present invention arecationic lipids. As used herein, the term “cationic lipid” is meant toinclude those lipids having one or two fatty acid or fatty alkyl chainsand an amino head group (including an alkylamino or dialkylamino group)that may be protonated to form a cationic lipid at physiological pH. Insome embodiments, a cationic lipid is referred to as an “amino lipid.”

Other cationic lipids would include those having alternative fatty acidgroups and other dialkylamino groups, including those in which the alkylsubstituents are different (e.g., N-ethyl-N-methylamino-,N-propyl-N-ethylamino- and the like). For those embodiments in which R₁and R₂ are both long chain alkyl or acyl groups, they can be the same ordifferent. In general, lipids (e.g., a cationic lipid) having lesssaturated acyl chains are more easily sized, particularly when thecomplexes are sized below about 0.3 microns, for purposes of filtersterilization. Cationic lipids containing unsaturated fatty acids withcarbon chain lengths in the range of C₁₀ to C₂₀ are typical. Otherscaffolds can also be used to separate the amino group (e.g., the aminogroup of the cationic lipid) and the fatty acid or fatty alkyl portionof the cationic lipid. Suitable scaffolds are known to those of skill inthe art.

In certain embodiments, cationic lipids of the present invention have atleast one protonatable or deprotonatable group, such that the lipid ispositively charged at a pH at or below physiological pH (e.g. pH 7.4),and neutral at a second pH, preferably at or above physiological pH.Such lipids are also referred to as cationic lipids. It will, of course,be understood that the addition or removal of protons as a function ofpH is an equilibrium process, and that the reference to a charged or aneutral lipid refers to the nature of the predominant species and doesnot require that all of the lipid be present in the charged or neutralform. Lipids that have more than one protonatable or deprotonatablegroup, or which are zwiterrionic, are not excluded from use in theinvention.

In certain embodiments, protonatable lipids (i.e., cationic lipids)according to the invention have a pKa of the protonatable group in therange of about 4 to about 11. Typically, lipids will have a pKa of about4 to about 7, e.g., between about 5 and 7, such as between about 5.5 and6.8, when incorporated into lipid particles. Such lipids will becationic at a lower pH formulation stage, while particles will belargely (though not completely) surface neutralized at physiological pHaround pH 7.4. One of the benefits of a pKa in the range of betweenabout 4 and 7 is that at least some nucleic acid associated with theoutside surface of the particle will lose its electrostatic interactionat physiological pH and be removed by simple dialysis; thus greatlyreducing the particle's susceptibility to clearance. pKa measurements oflipids within lipid particles can be performed, for example, by usingthe fluorescent probe 2-(p-toluidino)-6-napthalene sulfonic acid (TNS),using methods described in Cullis et al., (1986) Chem Phys Lipids 40,127-144.

In one embodiment, the formulations of the invention are entrapped by atleast 75%, at least 80% or at least 90%.

In one embodiment, the formulations of the invention further comprise anapolipoprotein. As used herein, the term “apolipoprotein” or“lipoprotein” refers to apolipoproteins known to those of skill in theart and variants and fragments thereof and to apolipoprotein agonists,analogues or fragments thereof described below. Suitable apolipoproteinsinclude, but are not limited to, ApoA-I, ApoA-II, ApoA-IV, ApoA-V andApoE, and active polymorphic forms, isoforms, variants and mutants aswell as fragments or truncated forms thereof. In certain embodiments,the apolipoprotein is a thiol containing apolipoprotein. “Thiolcontaining apolipoprotein” refers to an apolipoprotein, variant,fragment or isoform that contains at least one cysteine residue. Themost common thiol containing apolipoproteins are ApoA-I Milano(ApoA-I_(M)) and ApoA-I Paris (ApoA-I_(P)) which contain one cysteineresidue (Jia et al., 2002, Biochem. Biophys. Res. Comm. 297: 206-13;Bielicki and Oda, 2002, Biochemistry 41: 2089-96). ApoA-II, ApoE2 andApoE3 are also thiol containing apolipoproteins. Isolated ApoE and/oractive fragments and polypeptide analogues thereof, includingrecombinantly produced forms thereof, are described in U.S. Pat. Nos.5,672,685; 5,525,472; 5,473,039; 5,182,364; 5,177,189; 5,168,045;5,116,739; the disclosures of which are herein incorporated byreference. ApoE3 is disclosed in Weisgraber, et al., “Human E apoproteinheterogeneity: cysteine-arginine interchanges in the amino acid sequenceof the apo-E isoforms,” J. Biol. Chem. (1981) 256: 9077-9083; and Rall,et al., “Structural basis for receptor binding heterogeneity ofapolipoprotein E from type III hyperlipoproteinemic subjects,” Proc.Nat. Acad. Sci. (1982) 79: 4696-4700. See also GenBank accession numberK00396.

In certain embodiments, the apolipoprotein can be in its mature form, inits preproapolipoprotein form or in its proapolipoprotein form. Homo-and heterodimers (where feasible) of pro- and mature ApoA-I (Duverger etal., 1996, Arterioscler. Thromb. Vasc. Biol. 16(12):1424-29), ApoA-IMilano (Klon et al., 2000, Biophys. J. 79:(3)1679-87; Franceschini etal., 1985, J. Biol. Chem. 260: 1632-35), ApoA-I Paris (Daum et al.,1999, J. Mol. Med. 77:614-22), ApoA-II (Shelness et al., 1985, J. Biol.Chem. 260(14):8637-46; Shelness et al., 1984, J. Biol. Chem.259(15):9929-35), ApoA-IV (Duverger et al., 1991, Euro. J. Biochem.201(2):373-83), and ApoE (McLean et al., 1983, J. Biol. Chem.258(14):8993-9000) can also be utilized within the scope of theinvention.

In certain embodiments, the apolipoprotein can be a fragment, variant orisoform of the apolipoprotein. The term “fragment” refers to anyapolipoprotein having an amino acid sequence shorter than that of anative apolipoprotein and which fragment retains the activity of nativeapolipoprotein, including lipid binding properties. By “variant” ismeant substitutions or alterations in the amino acid sequences of theapolipoprotein, which substitutions or alterations, e.g., additions anddeletions of amino acid residues, do not abolish the activity of nativeapolipoprotein, including lipid binding properties. Thus, a variant cancomprise a protein or peptide having a substantially identical aminoacid sequence to a native apolipoprotein provided herein in which one ormore amino acid residues have been conservatively substituted withchemically similar amino acids. Examples of conservative substitutionsinclude the substitution of at least one hydrophobic residue such asisoleucine, valine, leucine or methionine for another. Likewise, thepresent invention contemplates, for example, the substitution of atleast one hydrophilic residue such as, for example, between arginine andlysine, between glutamine and asparagine, and between glycine and serine(see U.S. Pat. Nos. 6,004,925, 6,037,323 and 6,046,166). The term“isoform” refers to a protein having the same, greater or partialfunction and similar, identical or partial sequence, and may or may notbe the product of the same gene and usually tissue specific (seeWeisgraber 1990, J. Lipid Res. 31(8):1503-11; Hixson and Powers 1991, J.Lipid Res. 32(9):1529-35; Lackner et al., 1985, J. Biol. Chem.260(2):703-6; Hoeg et al., 1986, J. Biol. Chem. 261(9):3911-4; Gordon etal., 1984, J. Biol. Chem. 259(1):468-74; Powell et al., 1987, Cell50(6):831-40; Aviram et al., 1998, Arterioscler. Thromb. Vase. Biol.18(10):1617-24; Aviram et al., 1998, J. Clin. Invest. 101(8):1581-90;Billecke et al., 2000, Drug Metab. Dispos. 28(11):1335-42; Draganov etal., 2000, J. Biol. Chem. 275(43):33435-42; Steinmetz and Utermann 1985,J. Biol. Chem. 260(4):2258-64; Widler et al., 1980, J. Biol. Chem.255(21):10464-71; Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sacre etal., 2003, FEBS Lett. 540(1-3):181-7; Weers, et al., 2003, Biophys.Chem. 100(1-3):481-92; Gong et al., 2002, J. Biol. Chem.277(33):29919-26; Ohta et al., 1984, J. Biol. Chem. 259(23):14888-93 andU.S. Pat. No. 6,372,886).

In certain embodiments, the methods and compositions of the presentinvention include the use of a chimeric construction of anapolipoprotein. For example, a chimeric construction of anapolipoprotein can be comprised of an apolipoprotein domain with highlipid binding capacity associated with an apolipoprotein domaincontaining ischemia reperfusion protective properties. A chimericconstruction of an apolipoprotein can be a construction that includesseparate regions within an apolipoprotein (i.e., homologousconstruction) or a chimeric construction can be a construction thatincludes separate regions between different apolipoproteins (i.e.,heterologous constructions). Compositions comprising a chimericconstruction can also include segments that are apolipoprotein variantsor segments designed to have a specific character (e.g., lipid binding,receptor binding, enzymatic, enzyme activating, antioxidant orreduction-oxidation property) (see Weisgraber 1990, J. Lipid Res.31(8):1503-11; Hixson and Powers 1991, J. Lipid Res. 32(9):1529-35;Lackner et al., 1985, J. Biol. Chem. 260(2):703-6; Hoeg et al, 1986, J.Biol. Chem. 261(9):3911-4; Gordon et al., 1984, J. Biol. Chem.259(1):468-74; Powell et al., 1987, Cell 50(6):831-40; Aviram et al.,1998, Arterioscler. Thromb. Vasc. Biol. 18(10):1617-24; Aviram et al.,1998, J. Clin. Invest. 101(8):1581-90; Billecke et al., 2000, DrugMetab. Dispos. 28(11):1335-42; Draganov et al., 2000, J. Biol. Chem.275(43):33435-42; Steinmetz and Utermann 1985, J. Biol. Chem.260(4):2258-64; Widler et al., 1980, J. Biol. Chem. 255(21):10464-71;Dyer et al., 1995, J. Lipid Res. 36(1):80-8; Sorenson et al., 1999,Arterioscler. Thromb. Vasc. Biol. 19(9):2214-25; Palgunachari 1996,Arterioscler. Throb. Vasc. Biol. 16(2):328-38: Thurberg et al., J. Biol.Chem. 271(11):6062-70; Dyer 1991, J. Biol. Chem. 266(23):150009-15; Hill1998, J. Biol. Chem. 273(47):30979-84).

Apolipoproteins utilized in the invention also include recombinant,synthetic, semi-synthetic or purified apolipoproteins. Methods forobtaining apolipoproteins or equivalents thereof, utilized by theinvention are well-known in the art. For example, apolipoproteins can beseparated from plasma or natural products by, for example, densitygradient centrifugation or immunoaffinity chromatography, or producedsynthetically, semi-synthetically or using recombinant DNA techniquesknown to those of the art (see, e.g., Mulugeta et al., 1998, J.Chromatogr. 798(1-2): 83-90; Chung et al., 1980, J. Lipid Res.21(3):284-91; Cheung et al., 1987, J. Lipid Res. 28(8):913-29; Persson,et al., 1998, J. Chromatogr. 711:97-109; U.S. Pat. Nos. 5,059,528,5,834,596, 5,876,968 and 5,721,114; and PCT Publications WO 86/04920 andWO 87/02062).

Apolipoproteins utilized in the invention further include apolipoproteinagonists such as peptides and peptide analogues that mimic the activityof ApoA-I, ApoA-I Milano (ApoA-I_(M)), ApoA-I Paris (ApoA-I_(P)),ApoA-II, ApoA-IV, and ApoE. For example, the apolipoprotein can be anyof those described in U.S. Pat. Nos. 6,004,925, 6,037,323, 6,046,166,and 5,840,688, the contents of which are incorporated herein byreference in their entireties.

Apolipoprotein agonist peptides or peptide analogues can be synthesizedor manufactured using any technique for peptide synthesis known in theart including, e.g., the techniques described in U.S. Pat. Nos.6,004,925, 6,037,323 and 6,046,166. For example, the peptides may beprepared using the solid-phase synthetic technique initially describedby Merrifield (1963, J. Am. Chem. Soc. 85:2149-2154). Other peptidesynthesis techniques may be found in Bodanszky et al., PeptideSynthesis, John Wiley & Sons, 2d Ed., (1976) and other referencesreadily available to those skilled in the art. A summary of polypeptidesynthesis techniques can be found in Stuart and Young, Solid PhasePeptide. Synthesis, Pierce Chemical Company, Rockford, Ill., (1984).Peptides may also be synthesized by solution methods as described in TheProteins, Vol. II, 3d Ed., Neurath et. al., Eds., p. 105-237, AcademicPress, New York, N.Y. (1976). Appropriate protective groups for use indifferent peptide syntheses are described in the above-mentioned textsas well as in McOmie, Protective Groups in Organic Chemistry, PlenumPress, New York, N.Y. (1973). The peptides of the present inventionmight also be prepared by chemical or enzymatic cleavage from largerportions of, for example, apolipoprotein A-I.

In certain embodiments, the apolipoprotein can be a mixture ofapolipoproteins. In one embodiment, the apolipoprotein can be ahomogeneous mixture, that is, a single type of apolipoprotein. Inanother embodiment, the apolipoprotein can be a heterogeneous mixture ofapolipoproteins, that is, a mixture of two or more differentapolipoproteins. Embodiments of heterogenous mixtures of apolipoproteinscan comprise, for example, a mixture of an apolipoprotein from an animalsource and an apolipoprotein from a semi-synthetic source. In certainembodiments, a heterogenous mixture can comprise, for example, a mixtureof ApoA-I and ApoA-I Milano. In certain embodiments, a heterogeneousmixture can comprise, for example, a mixture of ApoA-I Milano and ApoA-IParis. Suitable mixtures for use in the methods and compositions of theinvention will be apparent to one of skill in the art.

If the apolipoprotein is obtained from natural sources, it can beobtained from a plant or animal source. If the apolipoprotein isobtained from an animal source, the apolipoprotein can be from anyspecies. In certain embodiments, the apolipoprotien can be obtained froman animal source. In certain embodiments, the apolipoprotein can beobtained from a human source. In preferred embodiments of the invention,the apolipoprotein is derived from the same species as the individual towhich the apolipoprotein is administered.

Lipid Particles

The present invention also provides lipid particles comprising one ormore of the cationic lipids described above. Lipid particles include,but are not limited to, liposomes. As used herein, a liposome is astructure having lipid-containing membranes enclosing an aqueousinterior. Liposomes may have one or more lipid membranes. The inventioncontemplates both single-layered liposomes, which are referred to asunilamellar, and multi-layered liposomes, which are referred to asmultilamellar. When complexed with nucleic acids, lipid particles mayalso be lipoplexes, which are composed of cationic lipid bilayerssandwiched between DNA layers, as described, e.g., in Felgner,Scientific American.

The lipid particles of the present invention may further comprise one ormore additional lipids and/or other components such as cholesterol.Other lipids may be included in the liposome compositions of the presentinvention for a variety of purposes, such as to prevent lipid oxidationor to attach ligands onto the liposome surface. Any of a number oflipids may be present in liposomes of the present invention, includingamphipathic, neutral, cationic, and anionic lipids. Such lipids can beused alone or in combination. Specific examples of additional lipidcomponents that may be present are described below.

Additional components that may be present in a lipid particle of thepresent invention include bilayer stabilizing components such aspolyamide oligomers (see, e.g., U.S. Pat. No. 6,320,017), peptides,proteins, detergents, lipid-derivatives, such as PEG coupled tophosphatidylethanolamine and PEG conjugated to ceramides (see, U.S. Pat.No. 5,885,613).

In particular embodiments, the lipid particles include one or more of asecond amino lipid or cationic lipid, a neutral lipid, a sterol, and alipid selected to reduce aggregation of lipid particles duringformation, which may result from steric stabilization of particles whichprevents charge-induced aggregation during formation.

Examples of lipids that reduce aggregation of particles during formationinclude polyethylene glycol (PEG)-modified lipids, monosialogangliosideGm1, and polyamide oligomers (“PAO”) such as (described in U.S. Pat. No.6,320,017). Other compounds with uncharged, hydrophilic, steric-barriermoieties, which prevent aggregation during formulation, like PEG, Gm1 orATTA, can also be coupled to lipids for use as in the methods andcompositions of the invention. ATTA-lipids are described, e.g., in U.S.Pat. No. 6,320,017, and PEG-lipid conjugates are described, e.g., inU.S. Pat. Nos. 5,820,873, 5,534,499 and 5,885,613. Typically, theconcentration of the lipid component selected to reduce aggregation isabout 1 to 15% (by mole percent of lipids).

Specific examples of PEG-modified lipids (or lipid-polyoxyethyleneconjugates) that are useful in the present invention can have a varietyof “anchoring” lipid portions to secure the PEG portion to the surfaceof the lipid vesicle. Examples of suitable PEG-modified lipids includePEG-modified phosphatidylethanolamine and phosphatidic acid,PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20) which aredescribed in co-pending U.S. Ser. No. 08/486,214, incorporated herein byreference, PEG-modified dialkylamines and PEG-modified1,2-diacyloxypropan-3-amines. Particularly preferred are PEG-modifieddiacylglycerols and dialkylglycerols.

In embodiments where a sterically-large moiety such as PEG or ATTA areconjugated to a lipid anchor, the selection of the lipid anchor dependson what type of association the conjugate is to have with the lipidparticle. It is well known that mPEG(mw2000)-diastearoylphosphatidylethanolamine (PEG-DSPE) will remainassociated with a liposome until the particle is cleared from thecirculation, possibly a matter of days. Other conjugates, such asPEG-CerC20 have similar staying capacity. PEG-CerC14, however, rapidlyexchanges out of the formulation upon exposure to serum, with a T_(1/2)less than 60 mins. in some assays. As illustrated in U.S. patentapplication Ser. No. 08/486,214, at least three characteristicsinfluence the rate of exchange: length of acyl chain, saturation of acylchain, and size of the steric-barrier head group. Compounds havingsuitable variations of these features may be useful for the invention.For some therapeutic applications it may be preferable for thePEG-modified lipid to be rapidly lost from the nucleic acid-lipidparticle in vivo and hence the PEG-modified lipid will possessrelatively short lipid anchors. In other therapeutic applications it maybe preferable for the nucleic acid-lipid particle to exhibit a longerplasma circulation lifetime and hence the PEG-modified lipid willpossess relatively longer lipid anchors.

It should be noted that aggregation preventing compounds do notnecessarily require lipid conjugation to function properly. Free PEG orfree ATTA in solution may be sufficient to prevent aggregation. If theparticles are stable after formulation, the PEG or ATTA can be dialyzedaway before administration to a subject.

Neutral lipids, when present in the lipid particle, can be any of anumber of lipid species which exist either in an uncharged or neutralzwitterionic form at physiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. Theselection of neutral lipids for use in the particles described herein isgenerally guided by consideration of, e.g., liposome size and stabilityof the liposomes in the bloodstream. Preferably, the neutral lipidcomponent is a lipid having two acyl groups, (i.e.,diacylphosphatidylcholine and diacylphosphatidylethanolamine). Lipidshaving a variety of acyl chain groups of varying chain length and degreeof saturation are available or may be isolated or synthesized bywell-known techniques. In one group of embodiments, lipids containingsaturated fatty acids with carbon chain lengths in the range of C₁₀ toC₂₀ are preferred. In another group of embodiments, lipids with mono ordiunsaturated fatty acids with carbon chain lengths in the range of C₁₀to C₂₀ are used. Additionally, lipids having mixtures of saturated andunsaturated fatty acid chains can be used. Preferably, the neutrallipids used in the present invention are DOPE, DSPC, POPC, DPPC or anyrelated phosphatidylcholine. The neutral lipids useful in the presentinvention may also be composed of sphingomyelin, dihydrosphingomyeline,or phospholipids with other head groups, such as serine and inositol.

The sterol component of the lipid mixture, when present, can be any ofthose sterols conventionally used in the field of liposome, lipidvesicle or lipid particle preparation. A preferred sterol ischolesterol.

Other cationic lipids, which carry a net positive charge at aboutphysiological pH, in addition to those specifically described above, mayalso be included in lipid particles of the present invention. Suchcationic lipids include, but are not limited to,N,N-dioleyl-N,N-dimethylammonium chloride (“DODAC”);N-(2,3-dioleyloxy)propyl-N,N—N-triethylammonium chloride (“DOTMA”);N,N-distearyl-N,N-dimethylammonium bromide (“DDAB”);N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”);1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“DOTAP.Cl”);3-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Choi”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine(“DOGS”), 1,2-dileoyl-sn-3-phosphoethanolamine (“DOPE”),1,2-dioleoyl-3-dimethylammonium propane (“DODAP”),N,N-dimethyl-2,3-dioleyloxy)propylamine (“DODMA”), andN-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations ofcationic lipids can be used, such as, e.g., LIPOFECTIN (including DOTMAand DOPE, available from GIBCO/BRL), and LIPOFECTAMINE (comprising DOSPAand DOPE, available from GIBCO/BRL). In particular embodiments, acationic lipid is an amino lipid.

Anionic lipids suitable for use in lipid particles of the presentinvention include, but are not limited to, phosphatidylglycerol,cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid,N-dodecanoyl phosphatidylethanoloamine, N-succinylphosphatidylethanolamine, N-glutaryl phosphatidylethanolamine,lysylphosphatidylglycerol, and other anionic modifying groups joined toneutral lipids.

In numerous embodiments, amphipathic lipids are included in lipidparticles of the present invention. “Amphipathic lipids” refer to anysuitable material, wherein the hydrophobic portion of the lipid materialorients into a hydrophobic phase, while the hydrophilic portion orientstoward the aqueous phase. Such compounds include, but are not limitedto, phospholipids, aminolipids, and sphingolipids. Representativephospholipids include sphingomyelin, phosphatidylcholine,phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol,phosphatidic acid, palmitoyloleoyl phosphatdylcholine,lysophosphatidylcholine, lysophosphatidylethanolamine,dipalmitoylphosphatidylcholine, dioleoylphosphatidylcholine,distearoylphosphatidylcholine, or dilinoleoylphosphatidylcholine. Otherphosphorus-lacking compounds, such as sphingolipids, glycosphingolipidfamilies, diacylglycerols, and -acyloxyacids, can also be used.Additionally, such amphipathic lipids can be readily mixed with otherlipids, such as triglycerides and sterols.

Also suitable for inclusion in the lipid particles of the presentinvention are programmable fusion lipids. Such lipid particles havelittle tendency to fuse with cell membranes and deliver their payloaduntil a given signal event occurs. This allows the lipid particle todistribute more evenly after injection into an organism or disease sitebefore it starts fusing with cells. The signal event can be, forexample, a change in pH, temperature, ionic environment, or time. In thelatter case, a fusion delaying or “cloaking” component, such as anATTA-lipid conjugate or a PEG-lipid conjugate, can simply exchange outof the lipid particle membrane over time. By the time the lipid particleis suitably distributed in the body, it has lost sufficient cloakingagent so as to be fusogenic. With other signal events, it is desirableto choose a signal that is associated with the disease site or targetcell, such as increased temperature at a site of inflammation.

In certain embodiments, it is desirable to target the lipid particles ofthis invention using targeting moieties that are specific to a cell typeor tissue. Targeting of lipid particles using a variety of targetingmoieties, such as ligands, cell surface receptors, glycoproteins,vitamins (e.g., riboflavin) and monoclonal antibodies, has beenpreviously described (see, e.g., U.S. Pat. Nos. 4,957,773 and4,603,044). The targeting moieties can comprise the entire protein orfragments thereof. Targeting mechanisms generally require that thetargeting agents be positioned on the surface of the lipid particle insuch a manner that the target moiety is available for interaction withthe target, for example, a cell surface receptor. A variety of differenttargeting agents and methods are known and available in the art,including those described, e.g., in Sapra, P. and Allen, T M, Prog.Lipid Res. 42(5):439-62 (2003); and Abra, R M et al., J. Liposome Res.12:1-3, (2002).

The use of lipid particles, i.e., liposomes, with a surface coating ofhydrophilic polymer chains, such as polyethylene glycol (PEG) chains,for targeting has been proposed (Allen, et al., Biochimica et BiophysicaActa 1237: 99-108 (1995); DeFrees, et al., Journal of the AmericanChemistry Society 118: 6101-6104 (1996); Blume, et al., Biochimica etBiophysica Acta 1149: 180-184 (1993); Klibanov, et al., Journal ofLiposome Research 2: 321-334 (1992); U.S. Pat. No. 5,013,556; Zalipsky,Bioconjugate Chemistry 4: 296-299 (1993); Zalipsky, FEBS Letters 353:71-74 (1994); Zalipsky, in Stealth Liposomes Chapter 9 (Lasic andMartin, Eds) CRC Press, Boca Raton Fl (1995). In one approach, a ligand,such as an antibody, for targeting the lipid particle is linked to thepolar head group of lipids forming the lipid particle. In anotherapproach, the targeting ligand is attached to the distal ends of the PEGchains forming the hydrophilic polymer coating (Klibanov, et al.,Journal of Liposome Research 2: 321-334 (1992); Kirpotin et al., FEBSLetters 388: 115-118 (1996)).

Standard methods for coupling the target agents can be used. Forexample, phosphatidylethanolamine, which can be activated for attachmentof target agents, or derivatized lipophilic compounds, such aslipid-derivatized bleomycin, can be used. Antibody-targeted liposomescan be constructed using, for instance, liposomes that incorporateprotein A (see, Renneisen, et al., J. Bio. Chem., 265:16337-16342 (1990)and Leonetti, et al., Proc. Natl. Acad. Sci. (USA), 87:2448-2451 (1990).Other examples of antibody conjugation are disclosed in U.S. Pat. No.6,027,726, the teachings of which are incorporated herein by reference.Examples of targeting moieties can also include other proteins, specificto cellular components, including antigens associated with neoplasms ortumors. Proteins used as targeting moieties can be attached to theliposomes via covalent bonds (see, Heath, Covalent Attachment ofProteins to Liposomes, 149 Methods in Enzymology 111-119 (AcademicPress, Inc. 1987)). Other targeting methods include the biotin-avidinsystem.

In one exemplary embodiment, the lipid particle comprises a mixture of acationic lipid of the present invention, neutral lipids (other than acationic lipid), a sterol (e.g., cholesterol) and a PEG-modified lipid(e.g., a PEG-DMG or PEG-DMA). In certain embodiments, the lipid mixtureconsists of or consists essentially of a cationic lipid of the presentinvention, a neutral lipid, cholesterol, and a PEG-modified lipid. Infurther preferred embodiments, the lipid particle consists of orconsists essentially of the above lipid mixture in molar ratios of about20-70% amino lipid: 5-45% neutral lipid:20-55% cholesterol:0.5-15%PEG-modified lipid.

In particular embodiments, the lipid particle consists of or consistsessentially of a cationic lipid chosen from Table 1 or Table 2, DSPC,Chol, and either PEG-DMG or PEG-DMA, e.g., in a molar ratio of about20-60% cationic lipid: 5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG orPEG-DMA. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA). In one embodiment, the cationic lipid is

In one preferred embodiment, the cationic lipid is

In another group of embodiments, the neutral lipid, DSPC, in thesecompositions is replaced with POPC, DPPC, DOPE or SM.

Therapeutic Agent-Lipid Particle Compositions and Formulations

The present invention includes compositions comprising a lipid particleof the present invention and an active agent, wherein the active agentis associated with the lipid particle. In particular embodiments, theactive agent is a therapeutic agent. In particular embodiments, theactive agent is encapsulated within an aqueous interior of the lipidparticle. In other embodiments, the active agent is present within oneor more lipid layers of the lipid particle. In other embodiments, theactive agent is bound to the exterior or interior lipid surface of alipid particle.

“Fully encapsulated” as used herein indicates that the nucleic acid inthe particles is not significantly degraded after exposure to serum or anuclease assay that would significantly degrade free nucleic acids. In afully encapsulated system, preferably less than 25% of particle nucleicacid is degraded in a treatment that would normally degrade 100% of freenucleic acid, more preferably less than 10% and most preferably lessthan 5% of the particle nucleic acid is degraded. Alternatively, fullencapsulation may be determined by an Oligreen® assay. Oligreen® is anultra-sensitive fluorescent nucleic acid stain for quantitatingoligonucleotides and single-stranded DNA in solution (available fromInvitrogen Corporation, Carlsbad, Calif.). Fully encapsulated alsosuggests that the particles are serum stable, that is, that they do notrapidly decompose into their component parts upon in vivoadministration.

Active agents, as used herein, include any molecule or compound capableof exerting a desired effect on a cell, tissue, organ, or subject. Sucheffects may be biological, physiological, or cosmetic, for example.Active agents may be any type of molecule or compound, including e.g.,nucleic acids, peptides and polypeptides, including, e.g., antibodies,such as, e.g., polyclonal antibodies, monoclonal antibodies, antibodyfragments; humanized antibodies, recombinant antibodies, recombinanthuman antibodies, and Primatized™ antibodies, cytokines, growth factors,apoptotic factors, differentiation-inducing factors, cell surfacereceptors and their ligands; hormones; and small molecules, includingsmall organic molecules or compounds.

In one embodiment, the active agent is a therapeutic agent, or a salt orderivative thereof. Therapeutic agent derivatives may be therapeuticallyactive themselves or they may be prodrugs, which become active uponfurther modification. Thus, in one embodiment, a therapeutic agentderivative retains some or all of the therapeutic activity as comparedto the unmodified agent, while in another embodiment, a therapeuticagent derivative lacks therapeutic activity.

In various embodiments, therapeutic agents include any therapeuticallyeffective agent or drug, such as anti-inflammatory compounds,anti-depressants, stimulants, analgesics, antibiotics, birth controlmedication, antipyretics, vasodilators, anti-angiogenics, cytovascularagents, signal transduction inhibitors, cardiovascular drugs, e.g.,anti-arrhythmic agents, vasoconstrictors, hormones, and steroids.

In certain embodiments, the therapeutic agent is an oncology drug, whichmay also be referred to as an anti-tumor drug, an anti-cancer drug, atumor drug, an antineoplastic agent, or the like. Examples of oncologydrugs that may be used according to the invention include, but are notlimited to, adriamycin, alkeran, allopurinol, altretamine, amifostine,anastrozole, araC, arsenic trioxide, azathioprine, bexarotene, biCNU,bleomycin, busulfan intravenous, busulfan oral, capecitabine (Xeloda),carboplatin, carmustine, CCNU, celecoxib, chlorambucil, cisplatin,cladribine, cyclosporin A, cytarabine, cytosine arabinoside,daunorubicin, cytoxan, daunorubicin, dexamethasone, dexrazoxane,dodetaxel, doxorubicin, doxorubicin, DTIC, epirubicin, estramustine,etoposide phosphate, etoposide and VP-16, exemestane, FK506,fludarabine, fluorouracil, 5-FU, gemcitabine (Gemzar),gemtuzumab-ozogamicin, goserelin acetate, hydrea, hydroxyurea,idarubicin, ifosfamide, imatinib mesylate, interferon, irinotecan(Camptostar, CPT-111), letrozole, leucovorin, leustatin, leuprolide,levamisole, litretinoin, megastrol, melphalan, L-PAM, mesna,methotrexate, methoxsalen, mithramycin, mitomycin, mitoxantrone,nitrogen mustard, paclitaxel, pamidronate, Pegademase, pentostatin,porfimer sodium, prednisone, rituxan, streptozocin, STI-571, tamoxifen,taxotere, temozolamide, teniposide, VM-26, topotecan (Hycamtin),toremifene, tretinoin, ATRA, valrubicin, velban, vinblastine,vincristine, VP16, and vinorelbine. Other examples of oncology drugsthat may be used according to the invention are ellipticin andellipticin analogs or derivatives, epothilones, intracellular kinaseinhibitors and camptothecins.

Nucleic Acid-Lipid Particles

In certain embodiments, lipid particles of the present invention areassociated with a nucleic acid, resulting in a nucleic acid-lipidparticle. In particular embodiments, the nucleic acid is fullyencapsulated in the lipid particle. As used herein, the term “nucleicacid” is meant to include any oligonucleotide or polynucleotide.Fragments containing up to 50 nucleotides are generally termedoligonucleotides, and longer fragments are called polynucleotides. Inparticular embodiments, oligonucletoides of the present invention are15-50 nucleotides in length.

In the context of this invention, the terms “polynucleotide” and“oligonucleotide” refer to a polymer or oligomer of nucleotide ornucleoside monomers consisting of naturally occurring bases, sugars andintersugar (backbone) linkages. The terms “polynucleotide” and“oligonucleotide” also includes polymers or oligomers comprisingnon-naturally occurring monomers, or portions thereof, which functionsimilarly. Such modified or substituted oligonucleotides are oftenpreferred over native forms because of properties such as, for example,enhanced cellular uptake and increased stability in the presence ofnucleases.

The nucleic acid that is present in a lipid-nucleic acid particleaccording to this invention includes any form of nucleic acid that isknown. The nucleic acids used herein can be single-stranded DNA or RNA,or double-stranded DNA or RNA, or DNA-RNA hybrids. Examples ofdouble-stranded DNA include structural genes, genes including controland termination regions, and self-replicating systems such as viral orplasmid DNA. Examples of double-stranded RNA include siRNA and other RNAinterference reagents. Single-stranded nucleic acids include, e.g.,antisense oligonucleotides, ribozymes, microRNA, and triplex-formingoligonucleotides. The nucleic acid that is present in a lipid-nucleicacid particle of this invention may include one or more of theoligonucleotide modifications described below.

Nucleic acids of the present invention may be of various lengths,generally dependent upon the particular form of nucleic acid. Forexample, in particular embodiments, plasmids or genes may be from about1,000 to 100,000 nucleotide residues in length. In particularembodiments, oligonucleotides may range from about 10 to 100 nucleotidesin length. In various related embodiments, oligonucleotides,single-stranded, double-stranded, and triple-stranded, may range inlength from about 10 to about 50 nucleotides, from about 20 o about 50nucleotides, from about 15 to about 30 nucleotides, from about 20 toabout 30 nucleotides in length.

In particular embodiments, the oligonucleotide (or a strand thereof) ofthe present invention specifically hybridizes to or is complementary toa target polynucleotide. “Specifically hybridizable” and “complementary”are terms which are used to indicate a sufficient degree ofcomplementarity such that stable and specific binding occurs between theDNA or RNA target and the oligonucleotide. It is understood that anoligonucleotide need not be 100% complementary to its target nucleicacid sequence to be specifically hybridizable. An oligonucleotide isspecifically hybridizable when binding of the oligonucleotide to thetarget interferes with the normal function of the target molecule tocause a loss of utility or expression therefrom, and there is asufficient degree of complementarity to avoid non-specific binding ofthe oligonucleotide to non-target sequences under conditions in whichspecific binding is desired, i.e., under physiological conditions in thecase of in vivo assays or therapeutic treatment, or, in the case of invitro assays, under conditions in which the assays are conducted. Thus,in other embodiments, this oligonucleotide includes 1, 2, or 3 basesubstitutions, e.g. mismatches, as compared to the region of a gene ormRNA sequence that it is targeting or to which it specificallyhybridizes.

RNA Interference Nucleic Acids

In particular embodiments, nucleic acid-lipid particles of the presentinvention are associated with RNA interference (RNAi) molecules. RNAinterference methods using RNAi molecules may be used to disrupt theexpression of a gene or polynucleotide of interest. Small interferingRNA (siRNA) has essentially replaced antisense ODN and ribozymes as thenext generation of targeted oligonucleotide drugs under development.

SiRNAs are RNA duplexes normally 16-30 nucleotides long that canassociate with a cytoplasmic multi-protein complex known as RNAi-inducedsilencing complex (RISC). RISC loaded with siRNA mediates thedegradation of homologous mRNA transcripts, therefore siRNA can bedesigned to knock down protein expression with high specificity. Unlikeother antisense technologies, siRNA function through a natural mechanismevolved to control gene expression through non-coding RNA. This isgenerally considered to be the reason why their activity is more potentin vitro and in vivo than either antisense ODN or ribozymes. A varietyof RNAi reagents, including siRNAs targeting clinically relevanttargets, are currently under pharmaceutical development, as described,e.g., in de Fougerolles, A. et al., Nature Reviews 6:443-453 (2007).

While the first described RNAi molecules were RNA:RNA hybrids comprisingboth an RNA sense and an RNA antisense strand, it has now beendemonstrated that DNA sense:RNA antisense hybrids, RNA sense:DNAantisense hybrids, and DNA:DNA hybrids are capable of mediating RNAi(Lamberton, J. S, and Christian, A. T., (2003) Molecular Biotechnology24:111-119). Thus, the invention includes the use of RNAi moleculescomprising any of these different types of double-stranded molecules. Inaddition, it is understood that RNAi molecules may be used andintroduced to cells in a variety of forms. Accordingly, as used herein,RNAi molecules encompasses any and all molecules capable of inducing anRNAi response in cells, including, but not limited to, double-strandedoligonucleotides comprising two separate strands, i.e. a sense strandand an antisense strand, e.g., small interfering RNA (siRNA);double-stranded oligonucleotide comprising two separate strands that arelinked together by non-nucleotidyl linker; oligonucleotides comprising ahairpin loop of complementary sequences, which forms a double-strandedregion, e.g., shRNAi molecules, and expression vectors that express oneor more polynucleotides capable of forming a double-strandedpolynucleotide alone or in combination with another polynucleotide.

A “single strand siRNA compound” as used herein, is an siRNA compoundwhich is made up of a single molecule. It may include a duplexed region,formed by intra-strand pairing, e.g., it may be, or include, a hairpinor pan-handle structure. Single strand siRNA compounds may be antisensewith regard to the target molecule

A single strand siRNA compound may be sufficiently long that it canenter the RISC and participate in RISC mediated cleavage of a targetmRNA. A single strand siRNA compound is at least 14, and in otherembodiments at least 15, 20, 25, 29, 35, 40, or 50 nucleotides inlength. In certain embodiments, it is less than 200, 100, or 60nucleotides in length.

Hairpin siRNA compounds will have a duplex region equal to or at least17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplexregion will may be equal to or less than 200, 100, or 50, in length. Incertain embodiments, ranges for the duplex region are 15-30, 17 to 23,19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin may havea single strand overhang or terminal unpaired region. In certainembodiments, the overhangs are 2-3 nucleotides in length. In someembodiments, the overhang is at the sense side of the hairpin and insome embodiments on the antisense side of the hairpin.

A “double stranded siRNA compound” as used herein, is an siRNA compoundwhich includes more than one, and in some cases two, strands in whichinterchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded siRNA compound may be equal toor at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides inlength. It may be equal to or less than 200, 100, or 50, nucleotides inlength. Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides inlength. As used herein, term “antisense strand” means the strand of ansiRNA compound that is sufficiently complementary to a target molecule,e.g. a target RNA.

The sense strand of a double stranded siRNA compound may be equal to orat least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length.It may be equal to or less than 200, 100, or 50, nucleotides in length.Ranges may be 17 to 25, 19 to 23, and 19 to 21 nucleotides in length.

The double strand portion of a double stranded siRNA compound may beequal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29,40, or 60 nucleotide pairs in length. It may be equal to or less than200, 100, or 50, nucleotides pairs in length. Ranges may be 15-30, 17 to23, 19 to 23, and 19 to 21 nucleotides pairs in length.

In many embodiments, the siRNA compound is sufficiently large that itcan be cleaved by an endogenous molecule, e.g., by Dicer, to producesmaller siRNA compounds, e.g., siRNAs agents

The sense and antisense strands may be chosen such that thedouble-stranded siRNA compound includes a single strand or unpairedregion at one or both ends of the molecule. Thus, a double-strandedsiRNA compound may contain sense and antisense strands, paired tocontain an overhang, e.g., one or two 5′ or 3′ overhangs, or a 3′overhang of 1-3 nucleotides. The overhangs can be the result of onestrand being longer than the other, or the result of two strands of thesame length being staggered. Some embodiments will have at least one 3′overhang. In one embodiment, both ends of an siRNA molecule will have a3′ overhang. In some embodiments, the overhang is 2 nucleotides.

In certain embodiments, the length for the duplexed region is between 15and 30, or 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., inthe ssiRNA compound range discussed above. ssiRNA compounds can resemblein length and structure the natural Dicer processed products from longdsiRNAs. Embodiments in which the two strands of the ssiRNA compound arelinked, e.g., covalently linked are also included. Hairpin, or othersingle strand structures which provide the required double strandedregion, and a 3′ overhang are also within the invention.

The siRNA compounds described herein, including double-stranded siRNAcompounds and single-stranded siRNA compounds can mediate silencing of atarget RNA, e.g., mRNA, e.g., a transcript of a gene that encodes aprotein. For convenience, such mRNA is also referred to herein as mRNAto be silenced. Such a gene is also referred to as a target gene. Ingeneral, the RNA to be silenced is an endogenous gene or a pathogengene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs,can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability tosilence, in a sequence specific manner, a target RNA. While not wishingto be bound by theory, it is believed that silencing uses the RNAimachinery or process and a guide RNA, e.g., an ssiRNA compound of 21 to23 nucleotides.

In one embodiment, an siRNA compound is “sufficiently complementary” toa target RNA, e.g., a target mRNA, such that the siRNA compound silencesproduction of protein encoded by the target mRNA. In another embodiment,the siRNA compound is “exactly complementary” to a target RNA, e.g., thetarget RNA and the siRNA compound anneal, for example to form a hybridmade exclusively of Watson-Crick base pairs in the region of exactcomplementarity. A “sufficiently complementary” target RNA can includean internal region (e.g., of at least 10 nucleotides) that is exactlycomplementary to a target RNA. Moreover, in certain embodiments, thesiRNA compound specifically discriminates a single-nucleotidedifference. In this case, the siRNA compound only mediates RNAi if exactcomplementary is found in the region (e.g., within 7 nucleotides of) thesingle-nucleotide difference.

MicroRNAs

Micro RNAs (miRNAs) are a highly conserved class of small RNA moleculesthat are transcribed from DNA in the genomes of plants and animals, butare not translated into protein. Processed miRNAs are single stranded˜17-25 nucleotide (nt) RNA molecules that become incorporated into theRNA-induced silencing complex (RISC) and have been identified as keyregulators of development, cell proliferation, apoptosis anddifferentiation. They are believed to play a role in regulation of geneexpression by binding to the 3′-untranslated region of specific mRNAs.RISC mediates down-regulation of gene expression through translationalinhibition, transcript cleavage, or both. RISC is also implicated intranscriptional silencing in the nucleus of a wide range of eukaryotes.

The number of miRNA sequences identified to date is large and growing,illustrative examples of which can be found, for example, in: “miRBase:microRNA sequences, targets and gene nomenclature” Griffiths-Jones S,Grocock R J, van Dongen S, Bateman A, Enright A J. NAR, 2006, 34,Database Issue, D140-D144; “The microRNA Registry” Griffiths-Jones S,NAR, 2004, 32, Database Issue, D109-D111; and also athttp://microrna.sanger.ac.uk/sequences/.

Antisense Oligonucleotides

In one embodiment, a nucleic acid is an antisense oligonucleotidedirected to a target polynucleotide. The term “antisenseoligonucleotide” or simply “antisense” is meant to includeoligonucleotides that are complementary to a targeted polynucleotidesequence. Antisense oligonucleotides are single strands of DNA or RNAthat are complementary to a chosen sequence, e.g. a target gene mRNA.Antisense oligonucleotides are thought to inhibit gene expression bybinding to a complementary mRNA. Binding to the target mRNA can lead toinhibition of gene expression by through making the

either by preventing translation of complementary mRNA strands bybinding to it or by leading to degradation of the target mRNA AntisenseDNA can be used to target a specific, complementary (coding ornon-coding) RNA. If binding takes places this DNA/RNA hybrid can bedegraded by the enzyme RNase H. In particular embodiment, antisenseoligonucleotides contain from about 10 to about 50 nucleotides, morepreferably about 15 to about 30 nucleotides. The term also encompassesantisense oligonucleotides that may not be exactly complementary to thedesired target gene. Thus, the invention can be utilized in instanceswhere non-target specific-activities are found with antisense, or wherean antisense sequence containing one or more mismatches with the targetsequence is the most preferred for a particular use.

Antisense oligonucleotides have been demonstrated to be effective andtargeted inhibitors of protein synthesis, and, consequently, can be usedto specifically inhibit protein synthesis by a targeted gene. Theefficacy of antisense oligonucleotides for inhibiting protein synthesisis well established. For example, the synthesis of polygalactauronaseand the muscarine type 2 acetylcholine receptor are inhibited byantisense oligonucleotides directed to their respective mRNA sequences(U.S. Pat. No. 5,739,119 and U.S. Pat. No. 5,759,829). Further, examplesof antisense inhibition have been demonstrated with the nuclear proteincyclin, the multiple drug resistance gene (MDG1), ICAM-1, E-selectin,STK-1, striatal GABA_(A) receptor and human EGF (Jaskulski et al.,Science. 1988 Jun. 10; 240(4858):1544-6; Vasanthakumar and Ahmed, CancerCommun. 1989; 1(4):225-32; Penis et al., Brain Res Mol Brain Res. 1998Jun. 15; 57(2):310-20; U.S. Pat. No. 5,801,154; U.S. Pat. No. 5,789,573;U.S. Pat. No. 5,718,709 and U.S. Pat. No. 5,610,288). Furthermore,antisense constructs have also been described that inhibit and can beused to treat a variety of abnormal cellular proliferations, e.g. cancer(U.S. Pat. No. 5,747,470; U.S. Pat. No. 5,591,317 and U.S. Pat. No.5,783,683).

Methods of producing antisense oligonucleotides are known in the art andcan be readily adapted to produce an antisense oligonucleotide thattargets any polynucleotide sequence. Selection of antisenseoligonucleotide sequences specific for a given target sequence is basedupon analysis of the chosen target sequence and determination ofsecondary structure, T_(m), binding energy, and relative stability.Antisense oligonucleotides may be selected based upon their relativeinability to form dimers, hairpins, or other secondary structures thatwould reduce or prohibit specific binding to the target mRNA in a hostcell. Highly preferred target regions of the mRNA include those regionsat or near the AUG translation initiation codon and those sequences thatare substantially complementary to 5′ regions of the mRNA. Thesesecondary structure analyses and target site selection considerationscan be performed, for example, using v.4 of the OLIGO primer analysissoftware (Molecular Biology Insights) and/or the BLASTN 2.0.5 algorithmsoftware (Altschul et al., Nucleic Acids Res. 1997, 25(17):3389-402).

Antagomirs

Antagomirs are RNA-like oligonucleotides that harbor variousmodifications for RNAse protection and pharmacologic properties, such asenhanced tissue and cellular uptake. They differ from normal RNA by, forexample, complete 2′-O-methylation of sugar, phosphorothioate backboneand, for example, a cholesterol-moiety at 3′-end. Antagomirs may be usedto efficiently silence endogenous miRNAs by forming duplexes comprisingthe antagomir and endogenous miRNA, thereby preventing miRNA-inducedgene silencing. An example of antagomir-mediated miRNA silencing is thesilencing of miR-122, described in Krutzfeldt et al, Nature, 2005, 438:685-689, which is expressly incorporated by reference herein in itsentirety. Antagomir RNAs may be synthesized using standard solid phaseoligonucleotide synthesis protocols. See U.S. patent application Ser.Nos. 11/502,158 and 11/657,341 (the disclosure of each of which areincorporated herein by reference).

An antagomir can include ligand-conjugated monomer subunits and monomersfor oligonucleotide synthesis. Exemplary monomers are described in U.S.application Ser. No. 10/916,185, filed on Aug. 10, 2004. An antagomircan have a ZXY structure, such as is described in PCT Application No.PCT/US2004/07070 filed on Mar. 8, 2004. An antagomir can be complexedwith an amphipathic moiety. Exemplary amphipathic moieties for use witholigonucleotide agents are described in PCT Application No.PCT/US2004/07070, filed on Mar. 8, 2004.

Aptamers

Aptamers are nucleic acid or peptide molecules that bind to a particularmolecule of interest with high affinity and specificity (Tuerk and Gold,Science 249:505 (1990); Ellington and Szostak, Nature 346:818 (1990)).DNA or RNA aptamers have been successfully produced which bind manydifferent entities from large proteins to small organic molecules. SeeEaton, Curr. Opin. Chem. Biol. 1:10-16 (1997), Famulok, Curr. Opin.Struct. Biol. 9:324-9 (1999), and Hermann and Patel, Science 287:820-5(2000). Aptamers may be RNA or DNA based, and may include a riboswitch.A riboswitch is a part of an mRNA molecule that can directly bind asmall target molecule, and whose binding of the target affects thegene's activity. Thus, an mRNA that contains a riboswitch is directlyinvolved in regulating its own activity, depending on the presence orabsence of its target molecule. Generally, aptamers are engineeredthrough repeated rounds of in vitro selection or equivalently, SELEX(systematic evolution of ligands by exponential enrichment) to bind tovarious molecular targets such as small molecules, proteins, nucleicacids, and even cells, tissues and organisms. The aptamer may beprepared by any known method, including synthetic, recombinant, andpurification methods, and may be used alone or in combination with otheraptamers specific for the same target. Further, as described more fullyherein, the term “aptamer” specifically includes “secondary aptamers”containing a consensus sequence derived from comparing two or more knownaptamers to a given target.

Ribozymes

According to another embodiment of the invention, nucleic acid-lipidparticles are associated with ribozymes. Ribozymes are RNA moleculescomplexes having specific catalytic domains that possess endonucleaseactivity (Kim and Cech, Proc Natl Acad Sci USA. 1987 December;84(24):8788-92; Forster and Symons, Cell. 1987 Apr. 24; 49(2):211-20).For example, a large number of ribozymes accelerate phosphoestertransfer reactions with a high degree of specificity, often cleavingonly one of several phosphoesters in an oligonucleotide substrate (Cechet al., Cell. 1981 December; 27(3 Pt 2):487-96; Michel and Westhof, JMol. Biol. 1990 Dec. 5; 216(3):585-610; Reinhold-Hurek and Shub, Nature.1992 May 14; 357(6374):173-6). This specificity has been attributed tothe requirement that the substrate bind via specific base-pairinginteractions to the internal guide sequence (“IGS”) of the ribozymeprior to chemical reaction.

At least six basic varieties of naturally-occurring enzymatic RNAs areknown presently. Each can catalyze the hydrolysis of RNA phosphodiesterbonds in trans (and thus can cleave other RNA molecules) underphysiological conditions. In general, enzymatic nucleic acids act byfirst binding to a target RNA. Such binding occurs through the targetbinding portion of a enzymatic nucleic acid which is held in closeproximity to an enzymatic portion of the molecule that acts to cleavethe target RNA. Thus, the enzymatic nucleic acid first recognizes andthen binds a target RNA through complementary base-pairing, and oncebound to the correct site, acts enzymatically to cut the target RNA.Strategic cleavage of such a target RNA will destroy its ability todirect synthesis of an encoded protein. After an enzymatic nucleic acidhas bound and cleaved its RNA target, it is released from that RNA tosearch for another target and can repeatedly bind and cleave newtargets.

The enzymatic nucleic acid molecule may be formed in a hammerhead,hairpin, a hepatitis δ virus, group I intron or RNaseP RNA (inassociation with an RNA guide sequence) or Neurospora VS RNA motif, forexample. Specific examples of hammerhead motifs are described by Rossiet al. Nucleic Acids Res. 1992 Sep. 11; 20(17):4559-65. Examples ofhairpin motifs are described by Hampel et al. (Eur. Pat. Appl. Publ. No.EP 0360257), Hampel and Tritz, Biochemistry 1989 Jun. 13;28(12):4929-33; Hampel et al., Nucleic Acids Res. 1990 Jan. 25;18(2):299-304 and U.S. Pat. No. 5,631,359. An example of the hepatitisvirus motif is described by Perrotta and Been, Biochemistry. 1992 Dec.1; 31(47):11843-52; an example of the RNaseP motif is described byGuerrier-Takada et al., Cell. 1983 December; 35(3 Pt 2):849-57;Neurospora VS RNA ribozyme motif is described by Collins (Saville andCollins, Cell. 1990 May 18; 61(4):685-96; Saville and Collins, Proc NatlAcad Sci USA. 1991 Oct. 1; 88(19):8826-30; Collins and Olive,Biochemistry. 1993 Mar. 23; 32(11):2795-9); and an example of the GroupI intron is described in U.S. Pat. No. 4,987,071. Importantcharacteristics of enzymatic nucleic acid molecules used according tothe invention are that they have a specific substrate binding site whichis complementary to one or more of the target gene DNA or RNA regions,and that they have nucleotide sequences within or surrounding thatsubstrate binding site which impart an RNA cleaving activity to themolecule. Thus the ribozyme constructs need not be limited to specificmotifs mentioned herein.

Methods of producing a ribozyme targeted to any polynucleotide sequenceare known in the art. Ribozymes may be designed as described in Int.Pat. Appl. Publ. No. WO 93/23569 and Int. Pat. Appl. Publ. No. WO94/02595, each specifically incorporated herein by reference, andsynthesized to be tested in vitro and in vivo, as described therein.

Ribozyme activity can be optimized by altering the length of theribozyme binding arms or chemically synthesizing ribozymes withmodifications that prevent their degradation by serum ribonucleases (seee.g., Int. Pat. Appl. Publ. No. WO 92/07065; Int. Pat. Appl. Publ. No.WO 93/15187; Int. Pat. Appl. Publ. No. WO 91/03162; Eur. Pat. Appl.Publ. No. 92110298.4; U.S. Pat. No. 5,334,711; and Int. Pat. Appl. Publ.No. WO 94/13688, which describe various chemical modifications that canbe made to the sugar moieties of enzymatic RNA molecules), modificationswhich enhance their efficacy in cells, and removal of stem II bases toshorten RNA synthesis times and reduce chemical requirements.

Immunostimulatory Oligonucleotides

Nucleic acids associated with lipid particles of the present inventionmay be immunostimulatory, including immunostimulatory oligonucleotides(ISS; single- or double-stranded) capable of inducing an immune responsewhen administered to a subject, which may be a mammal or other patient.ISS include, e.g., certain palindromes leading to hairpin secondarystructures (see Yamamoto S., et al. (1992) J. Immunol. 148: 4072-4076),or CpG motifs, as well as other known ISS features (such as multi-Gdomains, see WO 96/11266).

The immune response may be an innate or an adaptive immune response. Theimmune system is divided into a more innate immune system, and acquiredadaptive immune system of vertebrates, the latter of which is furtherdivided into humoral cellular components. In particular embodiments, theimmune response may be mucosal.

In particular embodiments, an immunostimulatory nucleic acid is onlyimmunostimulatory when administered in combination with a lipidparticle, and is not immunostimulatory when administered in its “freeform.” According to the present invention, such an oligonucleotide isconsidered to be immunostimulatory.

Immunostimulatory nucleic acids are considered to be non-sequencespecific when it is not required that they specifically bind to andreduce the expression of a target polynucleotide in order to provoke animmune response. Thus, certain immunostimulatory nucleic acids maycomprise a sequence corresponding to a region of a naturally occurringgene or mRNA, but they may still be considered non-sequence specificimmunostimulatory nucleic acids.

In one embodiment, the immunostimulatory nucleic acid or oligonucleotidecomprises at least one CpG dinucleotide. The oligonucleotide or CpGdinucleotide may be unmethylated or methylated. In another embodiment,the immunostimulatory nucleic acid comprises at least one CpGdinucleotide having a methylated cytosine. In one embodiment, thenucleic acid comprises a single CpG dinucleotide, wherein the cytosinein said CpG dinucleotide is methylated. In a specific embodiment, thenucleic acid comprises the sequence 5′ TAACGTTGAGGGGCAT 3′. In analternative embodiment, the nucleic acid comprises at least two CpGdinucleotides, wherein at least one cytosine in the CpG dinucleotides ismethylated. In a further embodiment, each cytosine in the CpGdinucleotides present in the sequence is methylated. In anotherembodiment, the nucleic acid comprises a plurality of CpG dinucleotides,wherein at least one of said CpG dinucleotides comprises a methylatedcytosine.

In one specific embodiment, the nucleic acid comprises the sequence 5′TTCCATGACGTTCCTGACGT 3′. In another specific embodiment, the nucleicacid sequence comprises the sequence 5′ TCCATGACGTTCCTGACGT 3′, whereinthe two cytosines indicated in bold are methylated. In particularembodiments, the ODN is selected from a group of ODNs consisting of ODN#1, ODN #2, ODN #3, ODN #4, ODN #5, ODN #6, ODN #7, ODN #8, and ODN #9,as shown below.

TABLE 3 Exemplary Immunostimulatory Oligonucleotides (ODNs) SEQ ODN NAMEID ODN SEQUENCE (5′-3′). ODN 1 5′-TAACGTTGAGGGGCAT-3 human c-myc* ODN 1m 5′-TAAZGTTGAGGGGCAT-3 ODN 2 5′-TCCATGACGTTCCTGACGTT-3 * ODN 2m5′-TCCATGAZGTTCCTGAZGTT-3 ODN 3 5′-TAAGCATACGGGGTGT-3 ODN 5 5′-AACGTT-3ODN 6 5′-GATGCTGTGTCGGGGTCTCCG GGC-3′ ODN 7 5′-TCGTCGTTTTGTCGTTTTGTCGTT-3′ ODN 7m 5′-TZGTZGTTTTGTZGTTTTGTZ GTT-3′ ODN 85′-TCCAGGACTTCTCTCAGGTT-3′ ODN 9 5′-TCTCCCAGCGTGCGCCAT-3′ ODN 10 murine5′-TGCATCCCCCAGGCCACCAT-3 Intracellular Adhesion Molecule-1 ODN 11 human5′-GCCCAAGCTGGCATCCGTCA-3′ Intracellular Adhesion Molecule-1ODN 12 human 5′-GCCCAAGCTGGCATCCGTCA-3′ IntracellularAdhesion Molecule-1 ODN 13 human erb-B-2 5′-GGT GCTCACTGC GGC-3′ODN 14 human c-myc 5′-AACC GTT GAG GGG CAT-3′ ODN 15 human c-myc5′-TAT GCT GTG CCG GGG TCT TCG GGC-3′ ODN 16 5′-GTGCCG GGGTCTTCGGGC-3′ODN 17 human Insulin 5′-GGACCCTCCTCCGGAGCC-3′ Growth Factor 1-ReceptorODN 18 human Insulin 5′-TCC TCC GGA GCC AGA Growth Factor CTT-3′1-Receptor ODN 19 human Epidermal 5′-AAC GTT GAG GGG CAT-3′Growth Factor-Receptor ODN 20 Epidermal 5′-CCGTGGTCA TGCTCC-3′ GrowthFactor-Receptor ODN 21 human Vascular 5′-CAG CCTGGCTCACCGEndothelial Growth CCTTGG-3′ Factor ODN 22 murine 5′-CAG CCA TGG TTC CCCPhosphokinase C-alpha CCA AC-3′ ODN 23 5′-GTT CTC GCT GGT GAG TTT CA-3′ODN 24 human Bc1-2 5′-TCT CCCAGCGTGCGCCAT-3′ ODN 25 human C-Raf-s5′-GTG CTC CAT TGA TGC-3′ ODN #26 human Vascular 5′- Endothelial GrowthGAGUUCUGAUGAGGCCGAAAGG- Factor Receptor-1 CCGAAAGUCUG-3′ ODN #275′-RRCGYY-3′ ODN #28 5′-AACGTTGAGGGGCAT-3′ ODN #295′-CAACGTTATGGGGAGA-3′ ODN #30 human c-myc 5′-TAACGTTGAGGGGCAT-3′ “Z”represents a methylated cytosine residue. ODN 14 is a 15-meroligonucleotide and ODN 1 is the same oligonucleotide having a thymidineadded onto the 5′ end making ODN 1 into a 16-mer. No difference inbiological activity between ODN 14 and ODN 1 has been detected and bothexhibit similar immunostimulatory activity (Mui et al., 2001)

Additional specific nucleic acid sequences of oligonucleotides (ODNs)suitable for use in the compositions and methods of the invention aredescribed in Raney et al., Journal of Pharmacology and ExperimentalTherapeutics, 298:1185-1192 (2001). In certain embodiments, ODNs used inthe compositions and methods of the present invention have aphosphodiester (“PO”) backbone or a phosphorothioate (“PS”) backbone,and/or at least one methylated cytosine residue in a CpG motif.

Decoy Oligonucleotides

Because transcription factors recognize their relatively short bindingsequences, even in the absence of surrounding genomic DNA, shortoligonucleotides bearing the consensus binding sequence of a specifictranscription factor can be used as tools for manipulating geneexpression in living cells. This strategy involves the intracellulardelivery of such “decoy oligonucleotides”, which are then recognized andbound by the target factor. Occupation of the transcription factor'sDNA-binding site by the decoy renders the transcription factor incapableof subsequently binding to the promoter regions of target genes. Decoyscan be used as therapeutic agents, either to inhibit the expression ofgenes that are activated by a transcription factor, or to upregulategenes that are suppressed by the binding of a transcription factor.Examples of the utilization of decoy oligonucleotides may be found inMann et al., J. Clin. Invest., 2000, 106: 1071-1075, which is expresslyincorporated by reference herein, in its entirety

Supermir

A supermir refers to a single stranded, double stranded or partiallydouble stranded oligomer or polymer of ribonucleic acid (RNA) ordeoxyribonucleic acid (DNA) or both or modifications thereof, which hasa nucleotide sequence that is substantially identical to an miRNA andthat is antisense with respect to its target. This term includesoligonucleotides composed of naturally-occurring nucleobases, sugars andcovalent internucleoside (backbone) linkages and which contain at leastone non-naturally-occurring portion which functions similarly. Suchmodified or substituted oligonucleotides are preferred over native formsbecause of desirable properties such as, for example, enhanced cellularuptake, enhanced affinity for nucleic acid target and increasedstability in the presence of nucleases. In a preferred embodiment, thesupermir does not include a sense strand, and in another preferredembodiment, the supermir does not self-hybridize to a significantextent. An supermir featured in the invention can have secondarystructure, but it is substantially single-stranded under physiologicalconditions. An supermir that is substantially single-stranded issingle-stranded to the extent that less than about 50% (e.g., less thanabout 40%, 30%, 20%, 10%, or 5%) of the supermir is duplexed withitself. The supermir can include a hairpin segment, e.g., sequence,preferably at the 3′ end can self hybridize and form a duplex region,e.g., a duplex region of at least 1, 2, 3, or 4 and preferably less than8, 7, 6, or n nucleotides, e.g., 5 nucleotides. The duplexed region canbe connected by a linker, e.g., a nucleotide linker, e.g., 3, 4, 5, or 6dTs, e.g., modified dTs. In another embodiment the supermir is duplexedwith a shorter oligo, e.g., of 5, 6, 7, 8, 9, or 10 nucleotides inlength, e.g., at one or both of the 3′ and 5′ end or at one end and inthe non-terminal or middle of the supermir.

miRNA Mimics

miRNA mimics represent a class of molecules that can be used to imitatethe gene silencing ability of one or more miRNAs. Thus, the term“microRNA mimic” refers to synthetic non-coding RNAs (i.e. the miRNA isnot obtained by purification from a source of the endogenous miRNA) thatare capable of entering the RNAi pathway and regulating gene expression.miRNA mimics can be designed as mature molecules (e.g. single stranded)or mimic precursors (e.g., pri- or pre-miRNAs) miRNA mimics can becomprised of nucleic acid (modified or modified nucleic acids) includingoligonucleotides comprising, without limitation, RNA, modified RNA, DNA,modified DNA, locked nucleic acids, or 2′-O,4′-C-ethylene-bridgednucleic acids (ENA), or any combination of the above (including DNA-RNAhybrids). In addition, miRNA mimics can comprise conjugates that canaffect delivery, intracellular compartmentalization, stability,specificity, functionality, strand usage, and/or potency. In one design,miRNA mimics are double stranded molecules (e.g., with a duplex regionof between about 16 and about 31 nucleotides in length) and contain oneor more sequences that have identity with the mature strand of a givenmiRNA. Modifications can comprise 2′ modifications (including 2′-Omethyl modifications and 2′ F modifications) on one or both strands ofthe molecule and internucleotide modifications (e.g. phorphorthioatemodifications) that enhance nucleic acid stability and/or specificity.In addition, miRNA mimics can include overhangs. The overhangs canconsist of 1-6 nucleotides on either the 3′ or 5′ end of either strandand can be modified to enhance stability or functionality. In oneembodiment, a miRNA mimic comprises a duplex region of between 16 and 31nucleotides and one or more of the following chemical modificationpatterns: the sense strand contains 2′-O-methyl modifications ofnucleotides 1 and 2 (counting from the 5′ end of the senseoligonucleotide), and all of the Cs and Us; the antisense strandmodifications can comprise 2′ F modification of all of the Cs and Us,phosphorylation of the 5′ end of the oligonucleotide, and stabilizedinternucleotide linkages associated with a 2 nucleotide 3′ overhang.

Antimir or miRNA Inhibitor.

The terms “antimir” “microRNA inhibitor”, “miR inhibitor”, or“inhibitor” are synonymous and refer to oligonucleotides or modifiedoligonucleotides that interfere with the ability of specific miRNAs. Ingeneral, the inhibitors are nucleic acid or modified nucleic acids innature including oligonucleotides comprising RNA, modified RNA, DNA,modified DNA, locked nucleic acids (LNAs), or any combination of theabove. Modifications include 2′ modifications (including 2′-0 alkylmodifications and 2′ F modifications) and internucleotide modifications(e.g. phosphorothioate modifications) that can affect delivery,stability, specificity, intracellular compartmentalization, or potency.In addition, miRNA inhibitors can comprise conjugates that can affectdelivery, intracellular compartmentalization, stability, and/or potency.Inhibitors can adopt a variety of configurations including singlestranded, double stranded (RNA/RNA or RNA/DNA duplexes), and hairpindesigns, in general, microRNA inhibitors comprise contain one or moresequences or portions of sequences that are complementary or partiallycomplementary with the mature strand (or strands) of the miRNA to betargeted, in addition, the miRNA inhibitor may also comprise additionalsequences located 5′ and 3′ to the sequence that is the reversecomplement of the mature miRNA. The additional sequences may be thereverse complements of the sequences that are adjacent to the maturemiRNA in the pri-miRNA from which the mature miRNA is derived, or theadditional sequences may be arbitrary sequences (having a mixture of A,G, C, or U). In some embodiments, one or both of the additionalsequences are arbitrary sequences capable of forming hairpins. Thus, insome embodiments, the sequence that is the reverse complement of themiRNA is flanked on the 5′ side and on the 3′ side by hairpinstructures. Micro-RNA inhibitors, when double stranded, may includemismatches between nucleotides on opposite strands. Furthermore,micro-RNA inhibitors may be linked to conjugate moieties in order tofacilitate uptake of the inhibitor into a cell. For example, a micro-RNAinhibitor may be linked to cholesteryl5-(bis(4-methoxyphenyl)(phenyl)methoxy)-3 hydroxypentylcarbamate) whichallows passive uptake of a micro-RNA inhibitor into a cell. Micro-RNAinhibitors, including hairpin miRNA inhibitors, are described in detailin Vermeulen et al., “Double-Stranded Regions Are Essential DesignComponents Of Potent Inhibitors of RISC Function,” RNA 13: 723-730(2007) and in WO2007/095387 and WO 2008/036825 each of which isincorporated herein by reference in its entirety. A person of ordinaryskill in the art can select a sequence from the database for a desiredmiRNA and design an inhibitor useful for the methods disclosed herein.

U1 Adaptor

U1 adaptor inhibit polyA sites and are bifunctional oligonucleotideswith a target domain complementary to a site in the target gene'sterminal exon and a ‘U1 domain’ that binds to the U1 smaller nuclear RNAcomponent of the U1 snRNP (Goraczniak, et al., 2008, NatureBiotechnology, 27(3), 257-263, which is expressly incorporated byreference herein, in its entirety). U1 snRNP is a ribonucleoproteincomplex that functions primarily to direct early steps in spliceosomeformation by binding to the pre-mRNA exon-intron boundary (Brown andSimpson, 1998, Annu Rev Plant Physiol Plant MoI Biol 49:77-95).Nucleotides 2-11 of the 5′end of U1 snRNA base pair bind with the 5′ssof the pre mRNA. In one embodiment, oligonucleotides of the inventionare U1 adaptors. In one embodiment, the U1 adaptor can be administeredin combination with at least one other iRNA agent.

Oligonucleotide Modifications

Unmodified oligonucleotides may be less than optimal in someapplications, e.g., unmodified oligonucleotides can be prone todegradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleicacid phosphodiester bonds. However, chemical modifications ofoligonucleotides can confer improved properties, and, e.g., can renderoligonucleotides more stable to nucleases.

As oligonucleotides are polymers of subunits or monomers, many of themodifications described below occur at a position which is repeatedwithin an oligonucleotide, e.g., a modification of a base, a sugar, aphosphate moiety, or the non-bridging oxygen of a phosphate moiety. Itis not necessary for all positions in a given oligonucleotide to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single oligonucleotide or even ata single nucleoside within an oligonucleotide.

In some cases the modification will occur at all of the subjectpositions in the oligonucleotide but in many, and in fact in most casesit will not. By way of example, a modification may only occur at a 3′ or5′ terminal position, may only occur in the internal region, may onlyoccur in a terminal regions, e.g. at a position on a terminal nucleotideor in the last 2, 3, 4, 5, or 10 nucleotides of an oligonucleotide. Amodification may occur in a double strand region, a single strandregion, or in both. A modification may occur only in the double strandregion of a double-stranded oligonucleotide or may only occur in asingle strand region of a double-stranded oligonucleotide. E.g., aphosphorothioate modification at a non-bridging oxygen position may onlyoccur at one or both termini, may only occur in a terminal regions,e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5,or 10 nucleotides of a strand, or may occur in double strand and singlestrand regions, particularly at termini. The 5′ end or ends can bephosphorylated.

A modification described herein may be the sole modification, or thesole type of modification included on multiple nucleotides, or amodification can be combined with one or more other modificationsdescribed herein. The modifications described herein can also becombined onto an oligonucleotide, e.g. different nucleotides of anoligonucleotide have different modifications described herein.

In some embodiments it is particularly preferred, e.g., to enhancestability, to include particular nucleobases in overhangs, or to includemodified nucleotides or nucleotide surrogates, in single strandoverhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can bedesirable to include purine nucleotides in overhangs. In someembodiments all or some of the bases in a 3′ or 5′ overhang will bemodified, e.g., with a modification described herein. Modifications caninclude, e.g., the use of modifications at the 2′ OH group of the ribosesugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine,instead of ribonucleotides, and modifications in the phosphate group,e.g., phosphothioate modifications. Overhangs need not be homologouswith the target sequence.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge isdistributed equally over the two non-bridging oxygen atoms. However, thephosphate group can be modified by replacing one of the oxygens with adifferent substituent. One result of this modification to RNA phosphatebackbones can be increased resistance of the oligoribonucleotide tonucleolytic breakdown. Thus while not wishing to be bound by theory, itcan be desirable in some embodiments to introduce alterations whichresult in either an uncharged linker or a charged linker withunsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate,phosphoroselenates, borano phosphates, borano phosphate esters, hydrogenphosphonates, phosphoroamidates, alkyl or aryl phosphonates andphosphotriesters. In certain embodiments, one of the non-bridgingphosphate oxygen atoms in the phosphate backbone moiety can be replacedby any of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl), C(i.e. an alkyl group, an aryl group, etc. . . . ), H, NR₂ (R ishydrogen, alkyl, aryl), or OR(R is alkyl or aryl). The phosphorous atomin an unmodified phosphate group is achiral. However, replacement of oneof the non-bridging oxygens with one of the above atoms or groups ofatoms renders the phosphorous atom chiral; in other words a phosphorousatom in a phosphate group modified in this way is a stereogenic center.The stereogenic phosphorous atom can possess either the “R”configuration (herein Rp) or the “S” configuration (herein Sp).

Phosphorodithioates have both non-bridging oxygens replaced by sulfur.The phosphorus center in the phosphorodithioates is achiral whichprecludes the formation of oligoribonucleotides diastereomers. Thus,while not wishing to be bound by theory, modifications to bothnon-bridging oxygens, which eliminate the chiral center, e.g.phosphorodithioate formation, may be desirable in that they cannotproduce diastereomer mixtures. Thus, the non-bridging oxygens can beindependently any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl).

The phosphate linker can also be modified by replacement of bridgingoxygen, (i.e. oxygen that links the phosphate to the nucleoside), withnitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates)and carbon (bridged methylenephosphonates). The replacement can occur atthe either linking oxygen or at both the linking oxygens. When thebridging oxygen is the 3′-oxygen of a nucleoside, replacement withcarbon is preferred. When the bridging oxygen is the 5′-oxygen of anucleoside, replacement with nitrogen is preferred.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containingconnectors. While not wishing to be bound by theory, it is believed thatsince the charged phosphodiester group is the reaction center innucleolytic degradation, its replacement with neutral structural mimicsshould impart enhanced nuclease stability. Again, while not wishing tobe bound by theory, it can be desirable, in some embodiment, tointroduce alterations in which the charged phosphate group is replacedby a neutral moiety.

Examples of moieties which can replace the phosphate group includemethyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl,carbamate, amide, thioether, ethylene oxide linker, sulfonate,sulfonamide, thioformacetal, formacetal, oxime, methyleneimino,methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo andmethyleneoxymethylimino. Preferred replacements include themethylenecarbonylamino and methylenemethylimino groups.

Modified phosphate linkages where at least one of the oxygens linked tothe phosphate has been replaced or the phosphate group has been replacedby a non-phosphorous group, are also referred to as “non phosphodiesterbackbone linkage.”

Replacement of Ribophosphate Backbone

Oligonucleotide-mimicking scaffolds can also be constructed wherein thephosphate linker and ribose sugar are replaced by nuclease resistantnucleoside or nucleotide surrogates. While not wishing to be bound bytheory, it is believed that the absence of a repetitively chargedbackbone diminishes binding to proteins that recognize polyanions (e.g.nucleases). Again, while not wishing to be bound by theory, it can bedesirable in some embodiment, to introduce alterations in which thebases are tethered by a neutral surrogate backbone. Examples include themophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA)nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Sugar Modifications

A modified RNA can include modification of all or some of the sugargroups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can bemodified or replaced with a number of different “oxy” or “deoxy”substituents. While not being bound by theory, enhanced stability isexpected since the hydroxyl can no longer be deprotonated to form a2′-alkoxide ion. The 2′-alkoxide can catalyze degradation byintramolecular nucleophilic attack on the linker phosphorus atom. Again,while not wishing to be bound by theory, it can be desirable to someembodiments to introduce alterations in which alkoxide formation at the2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy oraryloxy (OR, e.g., R=H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl orsugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked”nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by amethylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE(AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroaryl amino, or diheteroaryl amino, ethylene diamine,polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino).It is noteworthy that oligonucleotides containing only the methoxyethylgroup (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nucleasestabilities comparable to those modified with the robustphosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, whichare of particular relevance to the overhang portions of partially dsRNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroarylamino, or amino acid); NH(CH₂CH₂NKCH₂CH₂-AMINE (AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R(R=alkyl, cycloalkyl, aryl, aralkyl,heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; andalkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionallysubstituted with e.g., an amino functionality. Preferred substitutentsare 2′-methoxyethyl, 2′-OCH3, 2′-O-allyl, 2′-C-allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess theopposite stereochemical configuration than that of the correspondingcarbon in ribose. Thus, an oligonucleotide can include nucleotidescontaining e.g., arabinose, as the sugar. The monomer can have an alphalinkage at the 1′ position on the sugar, e.g., alpha-nucleosides.Oligonucleotides can also include “abasic” sugars, which lack anucleobase at C-1′. These abasic sugars can also be further containingmodifications at one or more of the constituent sugar atoms.Oligonucleotides can also contain one or more sugars that are in the Lform, e.g. L-nucleosides.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Suchmodifications can be at the 3′ end, 5′ end or both ends of the molecule.They can include modification or replacement of an entire terminalphosphate or of one or more of the atoms of the phosphate group. E.g.,the 3′ and 5′ ends of an oligonucleotide can be conjugated to otherfunctional molecular entities such as labeling moieties, e.g.,fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) orprotecting groups (based e.g., on sulfur, silicon, boron or ester). Thefunctional molecular entities can be attached to the sugar through aphosphate group and/or a linker. The terminal atom of the linker canconnect to or replace the linking atom of the phosphate group or theC-3′ or C-5′ 0, N, S or C group of the sugar. Alternatively, the linkercan connect to or replace the terminal atom of a nucleotide surrogate(e.g., PNAs).

When a linker/phosphate-functional molecular entity-linker/phosphatearray is interposed between two strands of a dsRNA, this array cansubstitute for a hairpin RNA loop in a hairpin-type RNA agent.

Terminal modifications useful for modulating activity includemodification of the 5′ end with phosphate or phosphate analogs. E.g., inpreferred embodiments antisense strands of dsRNAs, are 5′ phosphorylatedor include a phosphoryl analog at the 5′ prime terminus 5′-phosphatemodifications include those which are compatible with RISC mediated genesilencing. Suitable modifications include: 5′-monophosphate((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′);5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap(7-methylated or non-methylated)(7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap(Appp), and any modified or unmodified nucleotide cap structure(N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate(phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate(phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replacedmonophosphate, diphosphate and triphosphates (e.g.5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-).

Terminal modifications can also be useful for monitoring distribution,and in such cases the preferred groups to be added include fluorophores,e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminalmodifications can also be useful for enhancing uptake, usefulmodifications for this include cholesterol. Terminal modifications canalso be useful for cross-linking an RNA agent to another moiety;modifications useful for this include mitomycin C.

Nucleobases

Adenine, guanine, cytosine and uracil are the most common bases found inRNA. These bases can be modified or replaced to provide RNA's havingimproved properties. E.g., nuclease resistant oligoribonucleotides canbe prepared with these bases or with synthetic and natural nucleobases(e.g., inosine, thymine, xanthine, hypoxanthine, nubularine,isoguanisine, or tubercidine) and any one of the above modifications.Alternatively, substituted or modified analogs of any of the abovebases, e.g., “unusual bases”, “modified bases”, “non-natural bases” and“universal bases” described herein, can be employed. Examples includewithout limitation 2-aminoadenine, 6-methyl and other alkyl derivativesof adenine and guanine, 2-propyl and other alkyl derivatives of adenineand guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine,6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyluracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other8-substituted adenines and guanines, 5-trifluoromethyl and other5-substituted uracils and cytosines, 7-methylguanine, 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine,dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil,7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine,2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole,5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil,5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil,5-methylaminomethyl-2-thiouracil, 3-(3-amino-3-carboxypropyl)uracil,3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine,N6-methyladenine, N6-isopentyladenine,2-methylthio-N-6-isopentenyladenine, N-methylguanines, or O-alkylatedbases. Further purines and pyrimidines include those disclosed in U.S.Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia OfPolymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed.John Wiley & Sons, 1990, and those disclosed by Englisch et al.,Angewandte Chemie, International Edition, 1991, 30, 613.

Cationic Groups

Modifications to oligonucleotides can also include attachment of one ormore cationic groups to the sugar, base, and/or the phosphorus atom of aphosphate or modified phosphate backbone moiety. A cationic group can beattached to any atom capable of substitution on a natural, unusual oruniversal base. A preferred position is one that does not interfere withhybridization, i.e., does not interfere with the hydrogen bondinginteractions needed for base pairing. A cationic group can be attachede.g., through the C2′ position of a sugar or analogous position in acyclic or acyclic sugar surrogate. Cationic groups can include e.g.,protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino);aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino,dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂;alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino,heteroaryl amino, diheteroaryl amino, or amino acid); orNH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino,heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroarylamino).

Placement within an Oligonucleotide

Some modifications may preferably be included on an oligonucleotide at aparticular location, e.g., at an internal position of a strand, or onthe 5′ or 3′ end of an oligonucleotide. A preferred location of amodification on an oligonucleotide, may confer preferred properties onthe agent. For example, preferred locations of particular modificationsmay confer optimum gene silencing properties, or increased resistance toendonuclease or exonuclease activity.

One or more nucleotides of an oligonucleotide may have a 2′-5′ linkage.One or more nucleotides of an oligonucleotide may have invertedlinkages, e.g. 3′-3′, 5′-5′,2′-2′ or 2′-3′ linkages.

A double-stranded oligonucleotide may include at least one5′-uridine-adenine-3′ (5′-UA-3′) dinucleotide wherein the uridine is a2′-modified nucleotide, or a terminal 5′-uridine-guanine-3′ (5′-UG-3′)dinucleotide, wherein the 5′-uridine is a 2′-modified nucleotide, or aterminal 5′-cytidine-adenine-3′ (5′-CA-3′) dinucleotide, wherein the5′-cytidine is a 2′-modified nucleotide, or a terminal5′-uridine-uridine-3′ (5′-UU-3′) dinucleotide, wherein the 5′-uridine isa 2′-modified nucleotide, or a terminal 5′-cytidine-cytidine-3′(5′-CC-3′) dinucleotide, wherein the 5′-cytidine is a 2′-modifiednucleotide, or a terminal 5′-cytidine-uridine-3′ (5′-CU-3′)dinucleotide, wherein the 5′-cytidine is a 2′-modified nucleotide, or aterminal 5′-uridine-cytidine-3′ (5′-UC-3′) dinucleotide, wherein the5′-uridine is a 2′-modified nucleotide. Double-stranded oligonucleotidesincluding these modifications are particularly stabilized againstendonuclease activity.

General References

The oligoribonucleotides and oligoribonucleosides used in accordancewith this invention may be synthesized with solid phase synthesis, seefor example “Oligonucleotide synthesis, a practical approach”, Ed. M. J.Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A PracticalApproach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1,Modern machine-aided methods of oligodeoxyribonucleotide synthesis,Chapter 2, Oligoribonucleotide synthesis, Chapter3,2′-O—Methyloligoribonucleotide-s: synthesis and applications, Chapter4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis ofoligonucleotide phosphorodithioates, Chapter 6, Synthesis ofoligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7,Oligodeoxynucleotides containing modified bases. Other particularlyuseful synthetic procedures, reagents, blocking groups and reactionconditions are described in Martin, P., Helv. Chian. Acta, 1995, 78,486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48,2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49,6123-6194, or references referred to therein. Modification described inWO 00/44895, WO01/75164, or WO02/44321 can be used herein. Thedisclosure of all publications, patents, and published patentapplications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S.Pat. No. 5,508,270. The preparation of alkyl phosphonateoligoribonucleotides is described in U.S. Pat. No. 4,469,863. Thepreparation of phosphoramidite oligoribonucleotides is described in U.S.Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation ofphosphotriester oligoribonucleotides is described in U.S. Pat. No.5,023,243. The preparation of borano phosphate oligoribonucleotide isdescribed in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described inU.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonateoligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001,66, 2789-2801. Preparation of sulfur bridged nucleotides is described inSproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al.Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al.Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein.Specific modifications to the ribose can be found in the followingreferences: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36,831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938),“LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified hereinas MMI linked oligoribonucleosides, methylenedimethylhydrazo linkedoligoribonucleosides, also identified herein as MDH linkedoligoribonucleosides, and methylenecarbonylamino linkedoligonucleosides, also identified herein as amide-3 linkedoligoribonucleosides, and methyleneaminocarbonyl linkedoligonucleosides, also identified herein as amide-4 linkedoligoribonucleosides as well as mixed backbone compounds having, as forinstance, alternating MMI and PO or PS linkages can be prepared as isdescribed in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and inpublished PCT applications PCT/US92/04294 and PCT/US92/04305 (publishedas WO 92/20822 WO and 92/20823, respectively). Formacetal andthioformacetal linked oligoribonucleosides can be prepared as isdescribed in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxidelinked oligoribonucleosides can be prepared as is described in U.S. Pat.No. 5,223,618. Siloxane replacements are described in Cormier, J. F. etal. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements aredescribed in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethylreplacements are described in Edge, M. D. et al. J. Chem. Soc. PerkinTrans. 11972, 1991. Carbamate replacements are described in Stirchak, E.P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described inU.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared asis described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates canbe prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033,and other related patent disclosures. Peptide Nucleic Acids (PNAs) areknown per se and can be prepared in accordance with any of the variousprocedures referred to in Peptide Nucleic Acids (PNA): Synthesis,Properties and Potential Applications, Bioorganic & Medicinal Chemistry,1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat.No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisenseand Nucleic Acid Drug Development 12, 103-128 (2002) and referencestherein.

Nucleobases References

N-2 substituted purine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amiditescan be prepared as is described in U.S. Pat. No. 5,457,191.5,6-Substituted pyrimidine nucleoside amidites can be prepared as isdescribed in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleosideamidites can be prepared as is described in U.S. Pat. No. 5,484,908.

Linkers

The term “linker” means an organic moiety that connects two parts of acompound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR¹, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as substituted or unsubstituted alkyl, substitutedor unsubstituted alkenyl, substituted or unsubstituted alkynyl,arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl,heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl,heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl,cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl,alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl,alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl,alkylheteroarylalkyl, alkylheteroarylalkenyl, alkylheteroarylalkynyl,alkenylheteroarylalkyl, alkenylheteroarylalkenyl,alkenylheteroarylalkynyl, alkynylheteroarylalkyl,alkynylheteroarylalkenyl, alkynylheteroarylalkynyl,alkylheterocyclylalkyl, alkylheterocyclylalkenyl,alkylhererocyclylalkynyl, alkenylheterocyclylalkyl,alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl,alkynylheterocyclylalkyl, alkynylheterocyclylalkenyl,alkynylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl,alkylheteroaryl, alkenylheteroaryl, alkynylhereroaryl, where one or moremethylenes can be interrupted or terminated by O, S, S(O), SO₂, N(R¹)₂,C(O), cleavable linking group, substituted or unsubstituted aryl,substituted or unsubstituted heteroaryl, substituted or unsubstitutedheterocyclic; where R¹ is hydrogen, acyl, aliphatic or substitutedaliphatic.

In one embodiment, the linker is—[(P-Q-R)_(q)—X—(P′-Q′-R′)_(q′)]_(q″)-T-, wherein:

P, R, T, P′, R′ and T are each independently for each occurrence absent,CO, NH, O, S, OC(O), NHC(O), CH₂, CH₂NH, CH₂O; NHCH(R^(a))C(O),—C(O)—CH(R^(a))—NH—, CH═N—O,

or heterocyclyl;

Q and Q′ are each independently for each occurrence absent, —(CH₂)_(n)—,—C(R¹)(R²)(CH₂)_(n)—, —(CH₂)_(n)C(R¹)(R²)—, —(CH₂CH₂O)_(m)CH₂CH₂—, or—(CH₂CH₂O)_(m)CH₂CH₂NH—;

X is absent or a cleavable linking group;

R^(a) is H or an amino acid side chain;

R¹ and R² are each independently for each occurrence H, CH₃, OH, SH orN(R^(N))₂;

R^(N) is independently for each occurrence H, methyl, ethyl, propyl,isopropyl, butyl or benzyl;

q, q′ and q″ are each independently for each occurrence 0-20 and whereinthe repeating unit can be the same or different;

n is independently for each occurrence 1-20; and

m is independently for each occurrence 0-50.

In one embodiment, the linker comprises at least one cleavable linkinggroup.

In certain embodiments, the linker is a branched linker. The branchpointof the branched linker may be at least trivalent, but may be atetravalent, pentavalent or hexavalent atom, or a group presenting suchmultiple valencies. In certain embodiments, the branchpoint is, —N,—N(Q)-C, —O—C, —S—C, —SS—C, —C(O)N(Q)-C, —OC(O)N(Q)-C, —N(Q)C(O)—C, or—N(Q)C(O)O—C; wherein Q is independently for each occurrence H oroptionally substituted alkyl. In other embodiment, the branchpoint isglycerol or glycerol derivative.

Cleavable Linking Groups

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least 10 times or more,preferably at least 100 times faster in the target cell or under a firstreference condition (which can, e.g., be selected to mimic or representintracellular conditions) than in the blood of a subject, or under asecond reference condition (which can, e.g., be selected to mimic orrepresent conditions found in the blood or serum). Cleavable linkinggroups are susceptible to cleavage agents, e.g., pH, redox potential orthe presence of degradative molecules. Generally, cleavage agents aremore prevalent or found at higher levels or activities inside cells thanin serum or blood. Examples of such degradative agents include: redoxagents which are selected for particular substrates or which have nosubstrate specificity, including, e.g., oxidative or reductive enzymesor reductive agents such as mercaptans, present in cells, that candegrade a redox cleavable linking group by reduction; esterases;endosomes or agents that can create an acidic environment, e.g., thosethat result in a pH of five or lower; enzymes that can hydrolyze ordegrade an acid cleavable linking group by acting as a general acid,peptidases (which can be substrate specific), and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing the cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted. For example, livertargeting ligands can be linked to the cationic lipids through a linkerthat includes an ester group. Liver cells are rich in esterases, andtherefore the linker will be cleaved more efficiently in liver cellsthan in cell types that are not esterase-rich. Other cell-types rich inesterases include cells of the lung, renal cortex, and testis.

Linkers that contain peptide bonds can be used when targeting cell typesrich in peptidases, such as liver cells and synoviocytes.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus one can determine the relative susceptibility tocleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It may be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least 2, 4, 10 or 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood or serum (or under in vitro conditions selected to mimicextracellular conditions).

Redox Cleavable Linking Groups

One class of cleavable linking groups are redox cleavable linking groupsthat are cleaved upon reduction or oxidation. An example of reductivelycleavable linking group is a disulphide linking group (—S—S—). Todetermine if a candidate cleavable linking group is a suitable“reductively cleavable linking group,” or for example is suitable foruse with a particular iRNA moiety and particular targeting agent one canlook to methods described herein. For example, a candidate can beevaluated by incubation with dithiothreitol (DTT), or other reducingagent using reagents know in the art, which mimic the rate of cleavagewhich would be observed in a cell, e.g., a target cell. The candidatescan also be evaluated under conditions which are selected to mimic bloodor serum conditions. In a preferred embodiment, candidate compounds arecleaved by at most 10% in the blood. In preferred embodiments, usefulcandidate compounds are degraded at least 2, 4, 10 or 100 times fasterin the cell (or under in vitro conditions selected to mimicintracellular conditions) as compared to blood (or under in vitroconditions selected to mimic extracellular conditions). The rate ofcleavage of candidate compounds can be determined using standard enzymekinetics assays under conditions chosen to mimic intracellular media andcompared to conditions chosen to mimic extracellular media.

Phosphate-Based Cleavable Linking Groups

Phosphate-based cleavable linking groups are cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

Acid Cleavable Linking Groups

Acid cleavable linking groups are linking groups that are cleaved underacidic conditions. In preferred embodiments acid cleavable linkinggroups are cleaved in an acidic environment with a pH of about 6.5 orlower (e.g., about 6.0, 5.5, 5.0, or lower), or by agents such asenzymes that can act as a general acid. In a cell, specific low pHorganelles, such as endosomes and lysosomes can provide a cleavingenvironment for acid cleavable linking groups. Examples of acidcleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

Ester-Based Linking Groups

Ester-based cleavable linking groups are cleaved by enzymes such asesterases and amidases in cells. Examples of ester-based cleavablelinking groups include but are not limited to esters of alkylene,alkenylene and alkynylene groups. Ester cleavable linking groups havethe general formula —C(O)O—, or —OC(O)—. These candidates can beevaluated using methods analogous to those described above.

Peptide-Based Cleaving Groups

Peptide-based cleavable linking groups are cleaved by enzymes such aspeptidases and proteases in cells. Peptide-based cleavable linkinggroups are peptide bonds formed between amino acids to yieldoligopeptides (e.g., dipeptides, tripeptides etc.) and polypeptides.Peptide-based cleavable groups do not include the amide group(—C(O)NH—). The amide group can be formed between any alkylene,alkenylene or alkynelene. A peptide bond is a special type of amide bondformed between amino acids to yield peptides and proteins. The peptidebased cleavage group is generally limited to the peptide bond (i.e., theamide bond) formed between amino acids yielding peptides and proteinsand does not include the entire amide functional group. Peptide-basedcleavable linking groups have the general formula—NHCHR^(A)C(O)NHCHR^(B)C(O)—, where R^(A) and R^(B) are the R groups ofthe two adjacent amino acids. These candidates can be evaluated usingmethods analogous to those described above.

Ligands

A wide variety of entities can be coupled to the oligonucleotides andlipids of the present invention. Preferred moieties are ligands, whichare coupled, preferably covalently, either directly or indirectly via anintervening tether.

In preferred embodiments, a ligand alters the distribution, targeting orlifetime of the molecule into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Ligands providing enhancedaffinity for a selected target are also termed targeting ligands.Preferred ligands for conjugation to the lipids of the present inventionare targeting ligands.

Some ligands can have endosomolytic properties. The endosomolyticligands promote the lysis of the endosome and/or transport of thecomposition of the invention, or its components, from the endosome tothe cytoplasm of the cell. The endosomolytic ligand may be a polyanionicpeptide or peptidomimetic which shows pH-dependent membrane activity andfusogenicity. In certain embodiments, the endosomolytic ligand assumesits active conformation at endosomal pH. The “active” conformation isthat conformation in which the endosomolytic ligand promotes lysis ofthe endosome and/or transport of the composition of the invention, orits components, from the endosome to the cytoplasm of the cell.Exemplary endosomolytic ligands include the GALA peptide (Subbarao etal., Biochemistry, 1987, 26: 2964-2972), the EALA peptide (Vogel et al.,J. Am. Chem. Soc., 1996, 118: 1581-1586), and their derivatives (Turk etal., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments,the endosomolytic component may contain a chemical group (e.g., an aminoacid) which will undergo a change in charge or protonation in responseto a change in pH. The endosomolytic component may be linear orbranched. Exemplary primary sequences of peptide based endosomolyticligands are shown in Table 4.

TABLE 4 List of peptides with endosomolytic activity. NameSequence (N to C) Ref. GALA AALEALAEALEALAEALEALAEAAAAGGC 1 EALAAALAEALAEALAEALAEALAEALAAAAGGC 2 ALEALAEALEALAEA 3 INF-7GLFEAIEGFIENGWEGMIWDYG 4 Inf HA-2 GLFGAIAGFIENGWEGMIDGWYG 5 diINF-7GLF EAI EGFI ENGW EGMI DGWYGC 5 GLF EAI EGFI ENGW EGMI DGWYGC diINF3GLF EAI EGFI ENGW EGMI DGGC 6 GLF EAI EGFI ENGW EGMI DGGC GLFGLFGALAEALAEALAEHLAEALAEALEALAAGGSC 6 GALA-INF3GLFEAIEGFIENGWEGLAEALAEALEALAAGGSC 6 INF-5 GLF EAI EGFI ENGW EGnI DG K 4GLF EAI EGFI ENGW EGnI DG n, norleucine References 1. Subbarao et al.,Biochemistry, 1987, 26: 2964-2972. 2. Vogel et al., J. Am. Chem. Soc.,1996, 118: 1581-1586 3. Turk, M. J., Reddy, J. A. et al. (2002).Characterization of a novel pH-sensitive peptide that enhances drugrelease from folate-targeted liposomes at endosomal pHs. Biochim.Biophys. Acta 1559, 56-68. 4. Plank, C. Oberhauser, B. Mechtler, K.Koch, C. Wagner, E. (1994). The influence of endosome-disruptivepeptides on gene transfer using synthetic virus-like gene transfersystems, J. Biol. Chem. 269 12918-12924. 5. Mastrobattista, E., Koning,G. A. et al. (2002). Functional characterization of anendosome-disruptive peptide and its application in cytosolic delivery ofimmunoliposome-entrapped proteins. J. Biol. Chem. 277, 27135-43. 6.Oberhauser, B., Plank, C. et al. (1995). Enhancing endosomal exit ofnucleic acids using pH-sensitive viral fusion peptides. Deliv.Strategies Antisense Oligonucleotide Ther. 247-66.

Preferred ligands can improve transport, hybridization, and specificityproperties and may also improve nuclease resistance of the resultantnatural or modified oligoribonucleotide, or a polymeric moleculecomprising any combination of monomers described herein and/or naturalor modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., forenhancing uptake; diagnostic compounds or reporter groups e.g., formonitoring distribution; cross-linking agents; and nuclease-resistanceconferring moieties. General examples include lipids, steroids,vitamins, sugars, proteins, peptides, polyamines, and peptide mimics

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL),high-density lipoprotein (HDL), or globulin); an carbohydrate (e.g., adextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronicacid); or a lipid. The ligand may also be a recombinant or syntheticmolecule, such as a synthetic polymer, e.g., a synthetic polyamino acid,an oligonucleotide (e.g. an aptamer). Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, an RGD peptide, an RGDpeptide mimetic or an aptamer. Table 5 shows some examples of targetingligands and their associated receptors.

TABLE 5 Targeting Ligands and their associated receptors Liver CellsLigand Receptor 1) Parenchymal Galactose ASGP-R Cell (PC)(Asiologlycoprotein (Hepatocytes) receptor) Gal NAc ASPG-R(n-acetyl-galactosamine) Gal NAc Receptor Lactose Asialofetuin ASPG-r 2)Sinusoidal Hyaluronan Hyaluronan receptor Endothelial ProcollagenProcollagen receptor Cell (SEC) Negatively charged Scavenger receptorsmolecules Mannose Mannose receptors N-acetyl Glucosamine Scavengerreceptors Immunoglobulins Fc Receptor LPS CD14 Receptor Insulin Receptormediated transcytosis Transferrin Receptor mediated transcytosisAlbumins Non-specific Sugar-Albumin conjugates Mannose-6-phosphateMannose-6-phosphate receptor 3) Kupffer Mannose Mannose receptors Cell(KC) Fucose Fucose receptors Albumins Non-specific Mannose-albuminconjugates

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose, oraptamers. The ligand can be, for example, a lipopolysaccharide, anactivator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

The ligand can increase the uptake of the iRNA agent into the cell byactivating an inflammatory response, for example. Exemplary ligands thatwould have such an effect include tumor necrosis factor alpha(TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HAS, low density lipoprotein (LDL) andhigh-density lipoprotein (HDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The peptide or peptidomimetic moiety can be about 5-50 aminoacids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 aminoacids long (see Table 6, for example).

TABLE 6 Exemplary Cell Permeation Peptides. Cell Permeation PeptideAmino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKKDerossi et al., J. Biol. Chem. 269: 10444, 1994 Tat fragmentGRKKRRQRRRPPQC Vives et al., J. Biol. Chem., (48-60) 272: 16010, 1997Signal Sequence- GALFLGWLGAAGSTMGAWSQPKKKR Chaloin et al., Biochem.based peptide KV Biophys. Res. Commun., 243: 601, 1998 PVECLLIILRRRIRKQAHAHSK Elmquist et al., Exp. Cell Res., 269: 237, 2001Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga et al., FASEB J.,12: 67, 1998 Amphiphilic KLALKLALKALKAALKLA Oehlke et al., Mol. Ther.,model peptide 2: 339, 2000 Arg₉ RRRRRRRRRMitchell et al., J. Pept. Res., 56: 318, 2000 Bacterial cell wallKFFKFFKFFK permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFL RNLVPRTESCecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQG GPR α-defensinACYCRIPACIAGERRYGTCIYQGRLWA FCC b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYRGKAKCCK Bactenecin RKCRIVVIRVCR PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPPRFPPRFPGKR-NH2 Indolicidin ILPWKWPWWPWRR-NH2

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP. An RFGF analogue (e.g., amino acid sequenceAALLPVLLAAP) containing a hydrophobic MTS can also be a targetingmoiety. The peptide moiety can be a “delivery” peptide, which can carrylarge polar molecules including peptides, oligonucleotides, and proteinacross cell membranes. For example, sequences from the HIV Tat protein(GRKKRRQRRRPPQ) and the Drosophila Antennapedia protein(RQIKIWFQNRRMKWKK) have been found to be capable of functioning asdelivery peptides. A peptide or peptidomimetic can be encoded by arandom sequence of DNA, such as a peptide identified from aphage-display library, or one-bead-one-compound (OBOC) combinatoriallibrary (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide orpeptidomimetic tethered to an iRNA agent via an incorporated monomerunit is a cell targeting peptide such as an arginine-glycine-asparticacid (RGD)-peptide, or RGD mimic. A peptide moiety can range in lengthfrom about 5 amino acids to about 40 amino acids. The peptide moietiescan have a structural modification, such as to increase stability ordirect conformational properties. Any of the structural modificationsdescribed below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an iRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver an iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can beused. E.g., RGD containing peptides and peptidomimetics can targetcancer cells, in particular cells that exhibit an αvβ3 integrin. Thus,one could use RGD peptides, cyclic peptides containing RGD, RGD peptidesthat include D-amino acids, as well as synthetic RGD mimics. In additionto RGD, one can use other moieties that target the αvβ3 integrin ligand.Generally, such ligands can be used to control proliferating cells andangiogeneis. Preferred conjugates of this type lignads that targetsPECAM-1, VEGF, or other cancer gene, e.g., a cancer gene describedherein.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an iRNA agent and/orthe carrier oligomer can be an amphipathic α-helical peptide. Exemplaryamphipathic α-helical peptides include, but are not limited to,cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide(BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfishintestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2,dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides,esculentinis-1, and caerins. A number of factors will preferably beconsidered to maintain the integrity of helix stability. For example, amaximum number of helix stabilization residues will be utilized (e.g.,leu, ala, or lys), and a minimum number helix destabilization residueswill be utilized (e.g., proline, or cyclic monomeric units. The cappingresidue will be considered (for example Gly is an exemplary N-cappingresidue and/or C-terminal amidation can be used to provide an extraH-bond to stabilize the helix. Formation of salt bridges betweenresidues with opposite charges, separated by i±3, or i±4 positions canprovide stability. For example, cationic residues such as lysine,arginine, homo-arginine, ornithine or histidine can form salt bridgeswith the anionic residues glutamate or aspartate.

Peptide and peptidomimetic ligands include those having naturallyoccurring or modified peptides, e.g., D or L peptides; α, β, or γpeptides; N-methyl peptides; azapeptides; peptides having one or moreamide, i.e., peptide, linkages replaced with one or more urea, thiourea,carbamate, or sulfonyl urea linkages; or cyclic peptides.

The targeting ligand can be any ligand that is capable of targeting aspecific receptor. Examples are: folate, GalNAc, galactose, mannose,mannose-6P, clusters of sugars such as GalNAc cluster, mannose cluster,galactose cluster, or an apatamer. A cluster is a combination of two ormore sugar units. The targeting ligands also include integrin receptorligands, Chemokine receptor ligands, transferrin, biotin, serotoninreceptor ligands, PSMA, endothelin, GCPII, somatostatin, LDL and HDLligands. The ligands can also be based on nucleic acid, e.g., anaptamer. The aptamer can be unmodified or have any combination ofmodifications disclosed herein.

Endosomal release agents include imidazoles, poly or oligoimidazoles,PEIs, peptides, fusogenic peptides, polycarboxylates, polyacations,masked oligo or poly cations or anions, acetals, polyacetals,ketals/polyketyals, orthoesters, polymers with masked or unmaskedcationic or anionic charges, dendrimers with masked or unmasked cationicor anionic charges.

PK modulator stands for pharmacokinetic modulator. PK modulator includelipophiles, bile acids, steroids, phospholipid analogues, peptides,protein binding agents, PEG, vitamins etc. Examplary PK modulatorinclude, but are not limited to, cholesterol, fatty acids, cholic acid,lithocholic acid, dialkylglycerides, diacylglyceride, phospholipids,sphingolipids, naproxen, ibuprofen, vitamin E, biotin etc.Oligonucleotides that comprise a number of phosphorothioate linkages arealso known to bind to serum protein, thus short oligonucleotides, e.g.oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases,comprising multiple of phosphorothioate linkages in the backbone arealso amenable to the present invention as ligands (e.g. as PK modulatingligands).

In addition, aptamers that bind serum components (e.g. serum proteins)are also amenable to the present invention as PK modulating ligands.

Other ligands amenable to the invention are described in copendingapplications U.S. Ser. No. 10/916,185, filed Aug. 10, 2004; U.S. Ser.No. 10/946,873, filed Sep. 21, 2004; U.S. Ser. No. 10/833,934, filedAug. 3, 2007; U.S. Ser. No. 11/115,989 filed Apr. 27, 2005 and U.S. Ser.No. 11/944,227 filed Nov. 21, 2007, which are incorporated by referencein their entireties for all purposes.

When two or more ligands are present, the ligands can all have sameproperties, all have different properties or some ligands have the sameproperties while others have different properties. For example, a ligandcan have targeting properties, have endosomolytic activity or have PKmodulating properties. In a preferred embodiment, all the ligands havedifferent properties.

Ligands can be coupled to the oligonucleotides various places, forexample, 3′-end, 5′-end, and/or at an internal position. In preferredembodiments, the ligand is attached to the oligonucleotides via anintervening tether. The ligand or tethered ligand may be present on amonomer when said monomer is incorporated into the growing strand. Insome embodiments, the ligand may be incorporated via coupling to a“precursor” monomer after said “precursor” monomer has been incorporatedinto the growing strand. For example, a monomer having, e.g., anamino-terminated tether (i.e., having no associated ligand), e.g.,TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisensestrand. In a subsequent operation, i.e., after incorporation of theprecursor monomer into the strand, a ligand having an electrophilicgroup, e.g., a pentafluorophenyl ester or aldehyde group, cansubsequently be attached to the precursor monomer by coupling theelectrophilic group of the ligand with the terminal nucleophilic groupof the precursor monomer's tether.

For double-stranded oligonucleotides, ligands can be attached to one orboth strands. In some embodiments, a double-stranded iRNA agent containsa ligand conjugated to the sense strand. In other embodiments, adouble-stranded iRNA agent contains a ligand conjugated to the antisensestrand.

In some embodiments, ligand can be conjugated to nucleobases, sugarmoieties, or internucleosidic linkages of nucleic acid molecules.Conjugation to purine nucleobases or derivatives thereof can occur atany position including, endocyclic and exocyclic atoms. In someembodiments, the 2-, 6-, 7-, or 8-positions of a purine nucleobase areattached to a conjugate moiety. Conjugation to pyrimidine nucleobases orderivatives thereof can also occur at any position. In some embodiments,the 2-, 5-, and 6-positions of a pyrimidine nucleobase can besubstituted with a conjugate moiety. Conjugation to sugar moieties ofnucleosides can occur at any carbon atom. Example carbon atoms of asugar moiety that can be attached to a conjugate moiety include the 2′,3′, and 5′ carbon atoms. The 1′ position can also be attached to aconjugate moiety, such as in an abasic residue. Internucleosidiclinkages can also bear conjugate moieties. For phosphorus-containinglinkages (e.g., phosphodiester, phosphorothioate, phosphorodithiotate,phosphoroamidate, and the like), the conjugate moiety can be attacheddirectly to the phosphorus atom or to an O, N, or S atom bound to thephosphorus atom. For amine- or amide-containing internucleosidiclinkages (e.g., PNA), the conjugate moiety can be attached to thenitrogen atom of the amine or amide or to an adjacent carbon atom.

There are numerous methods for preparing conjugates of oligomericcompounds. Generally, an oligomeric compound is attached to a conjugatemoiety by contacting a reactive group (e.g., OH, SH, amine, carboxyl,aldehyde, and the like) on the oligomeric compound with a reactive groupon the conjugate moiety. In some embodiments, one reactive group iselectrophilic and the other is nucleophilic.

For example, an electrophilic group can be a carbonyl-containingfunctionality and a nucleophilic group can be an amine or thiol. Methodsfor conjugation of nucleic acids and related oligomeric compounds withand without linking groups are well described in the literature such as,for example, in Manoharan in Antisense Research and Applications, Crookeand LeBleu, eds., CRC Press, Boca Raton, Fla., 1993, Chapter 17, whichis incorporated herein by reference in its entirety.

Representative United States patents that teach the preparation ofoligonucleotide conjugates include, but are not limited to, U.S. Pat.Nos. 4,828,979; 4,948,882; 5,218, 105; 5,525,465; 5,541,313; 5,545,730;5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578, 718;5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737;4,824,941; 4,835,263; 4,876,335; 4,904, 582; 4,958,013; 5,082,830;5,112,963; 5,214,136; 5,082, 830; 5,112,963; 5,149,782; 5,214,136;5,245,022; 5,254, 469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510, 475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574, 142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,672,662;5,688,941; 5,714,166; 6,153, 737; 6,172,208; 6,300,319; 6,335,434;6,335,437; 6,395,437; 6,444,806; 6,486,308; 6,525,031; 6,528,631;6,559,279; each of which is herein incorporated by reference.

Characteristic of Nucleic Acid-Lipid Particles

In certain embodiments, the present invention relates to methods andcompositions for producing lipid-encapsulated nucleic acid particles inwhich nucleic acids are encapsulated within a lipid layer. Such nucleicacid-lipid particles, incorporating siRNA oligonucleotides, arecharacterized using a variety of biophysical parameters including: (1)drug to lipid ratio; (2) encapsulation efficiency; and (3) particlesize. High drug to lipid rations, high encapsulation efficiency, goodnuclease resistance and serum stability and controllable particle size,generally less than 200 nm in diameter are desirable. In addition, thenature of the nucleic acid polymer is of significance, since themodification of nucleic acids in an effort to impart nuclease resistanceadds to the cost of therapeutics while in many cases providing onlylimited resistance. Unless stated otherwise, these criteria arecalculated in this specification as follows:

Nucleic acid to lipid ratio is the amount of nucleic acid in a definedvolume of preparation divided by the amount of lipid in the same volume.This may be on a mole per mole basis or on a weight per weight basis, oron a weight per mole basis. For final, administration-readyformulations, the nucleic acid:lipid ratio is calculated after dialysis,chromatography and/or enzyme (e.g., nuclease) digestion has beenemployed to remove as much of the external nucleic acid as possible;

Encapsulation efficiency refers to the drug to lipid ratio of thestarting mixture divided by the drug to lipid ratio of the final,administration competent formulation. This is a measure of relativeefficiency. For a measure of absolute efficiency, the total amount ofnucleic acid added to the starting mixture that ends up in theadministration competent formulation, can also be calculated. The amountof lipid lost during the formulation process may also be calculated.Efficiency is a measure of the wastage and expense of the formulation;and

Size indicates the size (diameter) of the particles formed. Sizedistribution may be determined using quasi-elastic light scattering(QELS) on a Nicomp Model 370 sub-micron particle sizer. Particles under200 nm are preferred for distribution to neo-vascularized (leaky)tissues, such as neoplasms and sites of inflammation.

Pharmaceutical Compositions

The lipid particles of present invention, particularly when associatedwith a therapeutic agent, may beformulated as a pharmaceuticalcomposition, e.g., which further comprises a pharmaceutically acceptablediluent, excipient, or carrier, such as physiological saline orphosphate buffer, selected in accordance with the route ofadministration and standard pharmaceutical practice.

In particular embodiments, pharmaceutical compositions comprising thelipid-nucleic acid particles of the invention are prepared according tostandard techniques and further comprise a pharmaceutically acceptablecarrier. Generally, normal saline will be employed as thepharmaceutically acceptable carrier. Other suitable carriers include,e.g., water, buffered water, 0.9% saline, 0.3% glycine, and the like,including glycoproteins for enhanced stability, such as albumin,lipoprotein, globulin, etc. In compositions comprising saline or othersalt containing carriers, the carrier is preferably added followinglipid particle formation. Thus, after the lipid-nucleic acidcompositions are formed, the compositions can be diluted intopharmaceutically acceptable carriers such as normal saline.

The resulting pharmaceutical preparations may be sterilized byconventional, well known sterilization techniques. The aqueous solutionscan then be packaged for use or filtered under aseptic conditions andlyophilized, the lyophilized preparation being combined with a sterileaqueous solution prior to administration. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents and the like, for example, sodiumacetate, sodium lactate, sodium chloride, potassium chloride, calciumchloride, etc. Additionally, the lipidic suspension may includelipid-protective agents which protect lipids against free-radical andlipid-peroxidative damages on storage. Lipophilic free-radicalquenchers, such as α-tocopherol and water-soluble iron-specificchelators, such as ferrioxamine, are suitable.

The concentration of lipid particle or lipid-nucleic acid particle inthe pharmaceutical formulations can vary widely, i.e., from less thanabout 0.01%, usually at or at least about 0.05-5% to as much as 10 to30% by weight and will be selected primarily by fluid volumes,viscosities, etc., in accordance with the particular mode ofadministration selected. For example, the concentration may be increasedto lower the fluid load associated with treatment. This may beparticularly desirable in patients having atherosclerosis-associatedcongestive heart failure or severe hypertension. Alternatively,complexes composed of irritating lipids may be diluted to lowconcentrations to lessen inflammation at the site of administration. Inone group of embodiments, the nucleic acid will have an attached labeland will be used for diagnosis (by indicating the presence ofcomplementary nucleic acid). In this instance, the amount of complexesadministered will depend upon the particular label used, the diseasestate being diagnosed and the judgement of the clinician but willgenerally be between about 0.01 and about 50 mg per kilogram of bodyweight, preferably between about 0.1 and about 5 mg/kg of body weight.

As noted above, the lipid-therapeutic agent (e.g., nucleic acid)particles of the invention may include polyethylene glycol(PEG)-modified phospholipids, PEG-ceramide, or gangliosideG_(M1)-modified lipids or other lipids effective to prevent or limitaggregation. Addition of such components does not merely prevent complexaggregation. Rather, it may also provide a means for increasingcirculation lifetime and increasing the delivery of the lipid-nucleicacid composition to the target tissues.

The present invention also provides lipid-therapeutic agent compositionsin kit form. The kit will typically be comprised of a container that iscompartmentalized for holding the various elements of the kit. The kitwill contain the particles or pharmaceutical compositions of the presentinvention, preferably in dehydrated or concentrated form, withinstructions for their rehydration or dilution and administration. Incertain embodiments, the particles comprise the active agent, while inother embodiments, they do not.

Methods of Manufacture

The methods and compositions of the invention make use of certaincationic lipids, the synthesis, preparation and characterization ofwhich is described below and in the accompanying Examples. In addition,the present invention provides methods of preparing lipid particles,including those associated with a therapeutic agent, e.g., a nucleicacid. In the methods described herein, a mixture of lipids is combinedwith a buffered aqueous solution of nucleic acid to produce anintermediate mixture containing nucleic acid encapsulated in lipidparticles wherein the encapsulated nucleic acids are present in anucleic acid/lipid ratio of about 3 wt % to about 25 wt %, preferably 5to 15 wt %. The intermediate mixture may optionally be sized to obtainlipid-encapsulated nucleic acid particles wherein the lipid portions areunilamellar vesicles, preferably having a diameter of 30 to 150 nm, morepreferably about 40 to 90 nm. The pH is then raised to neutralize atleast a portion of the surface charges on the lipid-nucleic acidparticles, thus providing an at least partially surface-neutralizedlipid-encapsulated nucleic acid composition.

As described above, several of these cationic lipids are amino lipidsthat are charged at a pH below the pK_(a) of the amino group andsubstantially neutral at a pH above the pK_(a). These cationic lipidsare termed titratable cationic lipids and can be used in theformulations of the invention using a two-step process. First, lipidvesicles can be formed at the lower pH with titratable cationic lipidsand other vesicle components in the presence of nucleic acids. In thismanner, the vesicles will encapsulate and entrap the nucleic acids.Second, the surface charge of the newly formed vesicles can beneutralized by increasing the pH of the medium to a level above thepK_(a) of the titratable cationic lipids present, i.e., to physiologicalpH or higher. Particularly advantageous aspects of this process includeboth the facile removal of any surface adsorbed nucleic acid and aresultant nucleic acid delivery vehicle which has a neutral surface.Liposomes or lipid particles having a neutral surface are expected toavoid rapid clearance from circulation and to avoid certain toxicitieswhich are associated with cationic liposome preparations. Additionaldetails concerning these uses of such titratable cationic lipids in theformulation of nucleic acid-lipid particles are provided in U.S. Pat.No. 6,287,591 and U.S. Pat. No. 6,858,225, incorporated herein byreference.

It is further noted that the vesicles formed in this manner provideformulations of uniform vesicle size with high content of nucleic acids.Additionally, the vesicles have a size range of from about 30 to about150 nm, more preferably about 30 to about 90 nm.

Without intending to be bound by any particular theory, it is believedthat the very high efficiency of nucleic acid encapsulation is a resultof electrostatic interaction at low pH. At acidic pH (e.g. pH 4.0) thevesicle surface is charged and binds a portion of the nucleic acidsthrough electrostatic interactions. When the external acidic buffer isexchanged for a more neutral buffer (e.g., pH 7.5) the surface of thelipid particle or liposome is neutralized, allowing any external nucleicacid to be removed. More detailed information on the formulation processis provided in various publications (e.g., U.S. Pat. No. 6,287,591 andU.S. Pat. No. 6,858,225).

In view of the above, the present invention provides methods ofpreparing lipid/nucleic acid formulations. In the methods describedherein, a mixture of lipids is combined with a buffered aqueous solutionof nucleic acid to produce an intermediate mixture containing nucleicacid encapsulated in lipid particles, e.g., wherein the encapsulatednucleic acids are present in a nucleic acid/lipid ratio of about 10 wt %to about 20 wt %. The intermediate mixture may optionally be sized toobtain lipid-encapsulated nucleic acid particles wherein the lipidportions are unilamellar vesicles, preferably having a diameter of 30 to150 nm, more preferably about 40 to 90 nm. The pH is then raised toneutralize at least a portion of the surface charges on thelipid-nucleic acid particles, thus providing an at least partiallysurface-neutralized lipid-encapsulated nucleic acid composition.

In certain embodiments, the mixture of lipids includes at least twolipid components: a first lipid component of the present invention thatis selected from among lipids which have a pKa such that the lipid iscationic at pH below the pKa and neutral at pH above the pKa, and asecond lipid component that is selected from among lipids that preventparticle aggregation during lipid-nucleic acid particle formation. Inparticular embodiments, the amino lipid is a novel cationic lipid of thepresent invention.

In preparing the nucleic acid-lipid particles of the invention, themixture of lipids is typically a solution of lipids in an organicsolvent. This mixture of lipids can then be dried to form a thin film orlyophilized to form a powder before being hydrated with an aqueousbuffer to form liposomes. Alternatively, in a preferred method, thelipid mixture can be solubilized in a water miscible alcohol, such asethanol, and this ethanolic solution added to an aqueous bufferresulting in spontaneous liposome formation. In most embodiments, thealcohol is used in the form in which it is commercially available. Forexample, ethanol can be used as absolute ethanol (100%), or as 95%ethanol, the remainder being water. This method is described in moredetail in U.S. Pat. No. 5,976,567).

In one exemplary embodiment, the mixture of lipids is a mixture ofcationic lipids, neutral lipids (other than a cationic lipid), a sterol(e.g., cholesterol) and a PEG-modified lipid (e.g., a PEG-DMG orPEG-DMA) in an alcohol solvent. In preferred embodiments, the lipidmixture consists essentially of a cationic lipid, a neutral lipid,cholesterol and a PEG-modified lipid in alcohol, more preferablyethanol. In further preferred embodiments, the first solution consistsof the above lipid mixture in molar ratios of about 20-70% cationiclipid: 5-45% neutral lipid:20-55% cholesterol:0.5-15% PEG-modifiedlipid. In still further preferred embodiments, the first solutionconsists essentially of a lipid chosen from Table 1 or Table 2, DSPC,Chol and PEG-DMG or PEG-DMA, more preferably in a molar ratio of about20-60% cationic lipid: 5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG orPEG-DMA. In particular embodiments, the molar lipid ratio isapproximately 40/10/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA), 35/15/40/10 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA) or 52/13/30/5 (mol % cationic lipid/DSPC/Chol/PEG-DMG orPEG-DMA). In another group of preferred embodiments, the neutral lipidin these compositions is replaced with POPC, DPPC, DOPE or SM.

In accordance with the invention, the lipid mixture is combined with abuffered aqueous solution that may contain the nucleic acids. Thebuffered aqueous solution of is typically a solution in which the bufferhas a pH of less than the pK_(a) of the protonatable lipid in the lipidmixture. Examples of suitable buffers include citrate, phosphate,acetate, and MES. A particularly preferred buffer is citrate buffer.Preferred buffers will be in the range of 1-1000 mM of the anion,depending on the chemistry of the nucleic acid being encapsulated, andoptimization of buffer concentration may be significant to achievinghigh loading levels (see, e.g., U.S. Pat. No. 6,287,591 and U.S. Pat.No. 6,858,225). Alternatively, pure water acidified to pH 5-6 withchloride, sulfate or the like may be useful. In this case, it may besuitable to add 5% glucose, or another non-ionic solute which willbalance the osmotic potential across the particle membrane when theparticles are dialyzed to remove ethanol, increase the pH, or mixed witha pharmaceutically acceptable carrier such as normal saline. The amountof nucleic acid in buffer can vary, but will typically be from about0.01 mg/mL to about 200 mg/mL, more preferably from about 0.5 mg/mL toabout 50 mg/mL.

The mixture of lipids and the buffered aqueous solution of therapeuticnucleic acids is combined to provide an intermediate mixture. Theintermediate mixture is typically a mixture of lipid particles havingencapsulated nucleic acids. Additionally, the intermediate mixture mayalso contain some portion of nucleic acids which are attached to thesurface of the lipid particles (liposomes or lipid vesicles) due to theionic attraction of the negatively-charged nucleic acids andpositively-charged lipids on the lipid particle surface (the aminolipids or other lipid making up the protonatable first lipid componentare positively charged in a buffer having a pH of less than the pK_(a),of the protonatable group on the lipid). In one group of preferredembodiments, the mixture of lipids is an alcohol solution of lipids andthe volumes of each of the solutions is adjusted so that uponcombination, the resulting alcohol content is from about 20% by volumeto about 45% by volume. The method of combining the mixtures can includeany of a variety of processes, often depending upon the scale offormulation produced. For example, when the total volume is about 10-20mL or less, the solutions can be combined in a test tube and stirredtogether using a vortex mixer. Large-scale processes can be carried outin suitable production scale glassware.

Optionally, the lipid-encapsulated therapeutic agent (e.g., nucleicacid) complexes which are produced by combining the lipid mixture andthe buffered aqueous solution of therapeutic agents (nucleic acids) canbe sized to achieve a desired size range and relatively narrowdistribution of lipid particle sizes. Preferably, the compositionsprovided herein will be sized to a mean diameter of from about 70 toabout 200 nm, more preferably about 90 to about 130 nm. Severaltechniques are available for sizing liposomes to a desired size. Onesizing method is described in U.S. Pat. No. 4,737,323, incorporatedherein by reference. Sonicating a liposome suspension either by bath orprobe sonication produces a progressive size reduction down to smallunilamellar vesicles (SUVs) less than about 0.05 microns in size.Homogenization is another method which relies on shearing energy tofragment large liposomes into smaller ones. In a typical homogenizationprocedure, multilamellar vesicles are recirculated through a standardemulsion homogenizer until selected liposome sizes, typically betweenabout 0.1 and 0.5 microns, are observed. In both methods, the particlesize distribution can be monitored by conventional laser-beam particlesize determination. For certain methods herein, extrusion is used toobtain a uniform vesicle size.

Extrusion of liposome compositions through a small-pore polycarbonatemembrane or an asymmetric ceramic membrane results in a relativelywell-defined size distribution. Typically, the suspension is cycledthrough the membrane one or more times until the desired liposomecomplex size distribution is achieved. The liposomes may be extrudedthrough successively smaller-pore membranes, to achieve a gradualreduction in liposome size. In some instances, the lipid-nucleic acidcompositions which are formed can be used without any sizing.

In particular embodiments, methods of the present invention furthercomprise a step of neutralizing at least some of the surface charges onthe lipid portions of the lipid-nucleic acid compositions. By at leastpartially neutralizing the surface charges, unencapsulated nucleic acidis freed from the lipid particle surface and can be removed from thecomposition using conventional techniques. Preferably, unencapsulatedand surface adsorbed nucleic acids are removed from the resultingcompositions through exchange of buffer solutions. For example,replacement of a citrate buffer (pH about 4.0, used for forming thecompositions) with a HEPES-buffered saline (HBS pH about 7.5) solution,results in the neutralization of liposome surface and nucleic acidrelease from the surface. The released nucleic acid can then be removedvia chromatography using standard methods, and then switched into abuffer with a pH above the pKa of the lipid used.

Optionally the lipid vesicles (i.e., lipid particles) can be formed byhydration in an aqueous buffer and sized using any of the methodsdescribed above prior to addition of the nucleic acid. As describedabove, the aqueous buffer should be of a pH below the pKa of the aminolipid. A solution of the nucleic acids can then be added to these sized,preformed vesicles. To allow encapsulation of nucleic acids into such“pre-formed” vesicles the mixture should contain an alcohol, such asethanol. In the case of ethanol, it should be present at a concentrationof about 20% (w/w) to about 45% (w/w). In addition, it may be necessaryto warm the mixture of pre-formed vesicles and nucleic acid in theaqueous buffer-ethanol mixture to a temperature of about 25° C. to about50° C. depending on the composition of the lipid vesicles and the natureof the nucleic acid. It will be apparent to one of ordinary skill in theart that optimization of the encapsulation process to achieve a desiredlevel of nucleic acid in the lipid vesicles will require manipulation ofvariable such as ethanol concentration and temperature. Examples ofsuitable conditions for nucleic acid encapsulation are provided in theExamples. Once the nucleic acids are encapsulated within the prefromedvesicles, the external pH can be increased to at least partiallyneutralize the surface charge. Unencapsulated and surface adsorbednucleic acids can then be removed as described above.

Method of Use

The lipid particles of the present invention may be used to deliver atherapeutic agent to a cell, in vitro or in vivo. In particularembodiments, the therapeutic agent is a nucleic acid, which is deliveredto a cell using a nucleic acid-lipid particles of the present invention.While the following description o various methods of using the lipidparticles and related pharmaceutical compositions of the presentinvention are exemplified by description related to nucleic acid-lipidparticles, it is understood that these methods and compositions may bereadily adapted for the delivery of any therapeutic agent for thetreatment of any disease or disorder that would benefit from suchtreatment.

In certain embodiments, the present invention provides methods forintroducing a nucleic acid into a cell. Preferred nucleic acids forintroduction into cells are siRNA, immune-stimulating oligonucleotides,plasmids, antisense and ribozymes. These methods may be carried out bycontacting the particles or compositions of the present invention withthe cells for a period of time sufficient for intracellular delivery tooccur.

The compositions of the present invention can be adsorbed to almost anycell type. Once adsorbed, the nucleic acid-lipid particles can either beendocytosed by a portion of the cells, exchange lipids with cellmembranes, or fuse with the cells. Transfer or incorporation of thenucleic acid portion of the complex can take place via any one of thesepathways. Without intending to be limited with respect to the scope ofthe invention, it is believed that in the case of particles taken upinto the cell by endocytosis the particles then interact with theendosomal membrane, resulting in destabilization of the endosomalmembrane, possibly by the formation of non-bilayer phases, resulting inintroduction of the encapsulated nucleic acid into the cell cytoplasm.Similarly in the case of direct fusion of the particles with the cellplasma membrane, when fusion takes place, the liposome membrane isintegrated into the cell membrane and the contents of the liposomecombine with the intracellular fluid. Contact between the cells and thelipid-nucleic acid compositions, when carried out in vitro, will takeplace in a biologically compatible medium. The concentration ofcompositions can vary widely depending on the particular application,but is generally between about 1 μmol and about 10 mmol. In certainembodiments, treatment of the cells with the lipid-nucleic acidcompositions will generally be carried out at physiological temperatures(about 37° C.) for periods of time from about 1 to 24 hours, preferablyfrom about 2 to 8 hours. For in vitro applications, the delivery ofnucleic acids can be to any cell grown in culture, whether of plant oranimal origin, vertebrate or invertebrate, and of any tissue or type. Inpreferred embodiments, the cells will be animal cells, more preferablymammalian cells, and most preferably human cells.

In one group of embodiments, a lipid-nucleic acid particle suspension isadded to 60-80% confluent plated cells having a cell density of fromabout 10³ to about 10⁵ cells/mL, more preferably about 2×10⁴ cells/mL.The concentration of the suspension added to the cells is preferably offrom about 0.01 to 20 μg/mL, more preferably about 1 μg/mL.

In another embodiment, the lipid particles of the invention can be maybe used to deliver a nucleic acid to a cell or cell line (for example, atumor cell line). Non-limiting examples of such cell lines include: HELA(ATCC Cat N: CCL-2), KB (ATCC Cat N: CCL-17), HEP3B (ATCC Cat N:HB-8064), SKOV-3 (ATCC Cat N: HTB-77), HCT-116 (ATCC Cat N: CCL-247),HT-29 (ATCC Cat N: HTB-38), PC-3 (ATCC Cat N: CRL-1435), A549 (ATCC CatN: CCL-185), MDA-MB-231 (ATCC Cat N: HTB-26).

Typical applications include using well known procedures to provideintracellular delivery of siRNA to knock down or silence specificcellular targets. Alternatively applications include delivery of DNA ormRNA sequences that code for therapeutically useful polypeptides. Inthis manner, therapy is provided for genetic diseases by supplyingdeficient or absent gene products (i.e., for Duchenne's dystrophy, seeKunkel, et al., Brit. Med. Bull. 45(3):630-643 (1989), and for cysticfibrosis, see Goodfellow, Nature 341:102-103 (1989)). Other uses for thecompositions of the present invention include introduction of antisenseoligonucleotides in cells (see, Bennett, et al., Mol. Pharm.41:1023-1033 (1992)).

Alternatively, the compositions of the present invention can also beused for deliver of nucleic acids to cells in vivo, using methods whichare known to those of skill in the art. With respect to delivery of DNAor mRNA sequences, Zhu, et al., Science 261:209-211 (1993), incorporatedherein by reference, describes the intravenous delivery ofcytomegalovirus (CMV)-chloramphenicol acetyltransferase (CAT) expressionplasmid using DOTMA-DOPE complexes. Hyde, et al., Nature 362:250-256(1993), incorporated herein by reference, describes the delivery of thecystic fibrosis transmembrane conductance regulator (CFTR) gene toepithelia of the airway and to alveoli in the lung of mice, usingliposomes. Brigham, et al., Am. J. Med. Sci. 298:278-281 (1989),incorporated herein by reference, describes the in vivo transfection oflungs of mice with a functioning prokaryotic gene encoding theintracellular enzyme, chloramphenicol acetyltransferase (CAT). Thus, thecompositions of the invention can be used in the treatment of infectiousdiseases.

For in vivo administration, the pharmaceutical compositions arepreferably administered parenterally, i.e., intraarticularly,intravenously, intraperitoneally, subcutaneously, or intramuscularly. Inparticular embodiments, the pharmaceutical compositions are administeredintravenously or intraperitoneally by a bolus injection. For oneexample, see Stadler, et al., U.S. Pat. No. 5,286,634, which isincorporated herein by reference. Intracellular nucleic acid deliveryhas also been discussed in Straubringer, et al., METHODS IN ENZYMOLOGY,Academic Press, New York. 101:512-527 (1983); Mannino, et al.,Biotechniques 6:682-690 (1988); Nicolau, et al., Crit. Rev. Ther. DrugCarrier Syst. 6:239-271 (1989), and Behr, Acc. Chem. Res. 26:274-278(1993). Still other methods of administering lipid-based therapeuticsare described in, for example, Rahman et al., U.S. Pat. No. 3,993,754;Sears, U.S. Pat. No. 4,145,410; Papahadjopoulos et al., U.S. Pat. No.4,235,871; Schneider, U.S. Pat. No. 4,224,179; Lenk et al., U.S. Pat.No. 4,522,803; and Fountain et al., U.S. Pat. No. 4,588,578.

In other methods, the pharmaceutical preparations may be contacted withthe target tissue by direct application of the preparation to thetissue. The application may be made by topical, “open” or “closed”procedures. By “topical,” it is meant the direct application of thepharmaceutical preparation to a tissue exposed to the environment, suchas the skin, oropharynx, external auditory canal, and the like. “Open”procedures are those procedures which include incising the skin of apatient and directly visualizing the underlying tissue to which thepharmaceutical preparations are applied. This is generally accomplishedby a surgical procedure, such as a thoracotomy to access the lungs,abdominal laparotomy to access abdominal viscera, or other directsurgical approach to the target tissue. “Closed” procedures are invasiveprocedures in which the internal target tissues are not directlyvisualized, but accessed via inserting instruments through small woundsin the skin. For example, the preparations may be administered to theperitoneum by needle lavage. Likewise, the pharmaceutical preparationsmay be administered to the meninges or spinal cord by infusion during alumbar puncture followed by appropriate positioning of the patient ascommonly practiced for spinal anesthesia or metrazamide imaging of thespinal cord. Alternatively, the preparations may be administered throughendoscopic devices.

The lipid-nucleic acid compositions can also be administered in anaerosol inhaled into the lungs (see, Brigham, et al., Am. J. Sci.298(4):278-281 (1989)) or by direct injection at the site of disease(Culver, Human Gene Therapy, MaryAnn Liebert, Inc., Publishers, NewYork. pp. 70-71 (1994)).

The methods of the present invention may be practiced in a variety ofhosts. Preferred hosts include mammalian species, such as humans,non-human primates, dogs, cats, cattle, horses, sheep, and the like.

Dosages for the lipid-therapeutic agent particles of the presentinvention will depend on the ratio of therapeutic agent to lipid and theadministrating physician's opinion based on age, weight, and conditionof the patient.

In one embodiment, the present invention provides a method of modulatingthe expression of a target polynucleotide or polypeptide. These methodsgenerally comprise contacting a cell with a lipid particle of thepresent invention that is associated with a nucleic acid capable ofmodulating the expression of a target polynucleotide or polypeptide. Asused herein, the term “modulating” refers to altering the expression ofa target polynucleotide or polypeptide. In different embodiments,modulating can mean increasing or enhancing, or it can mean decreasingor reducing. Methods of measuring the level of expression of a targetpolynucleotide or polypeptide are known and available in the arts andinclude, e.g., methods employing reverse transcription-polymerase chainreaction (RT-PCR) and immunohistochemical techniques. In particularembodiments, the level of expression of a target polynucleotide orpolypeptide is increased or reduced by at least 10%, 20%, 30%, 40%, 50%,or greater than 50% as compared to an appropriate control value.

For example, if increased expression of a polypeptide desired, thenucleic acid may be an expression vector that includes a polynucleotidethat encodes the desired polypeptide. On the other hand, if reducedexpression of a polynucleotide or polypeptide is desired, then thenucleic acid may be, e.g., an antisense oligonucleotide, siRNA, ormicroRNA that comprises a polynucleotide sequence that specificallyhybridizes to a polnucleotide that encodes the target polypeptide,thereby disrupting expression of the target polynucleotide orpolypeptide. Alternatively, the nucleic acid may be a plasmid thatexpresses such an antisense oligonucletoide, siRNA, or microRNA.

In one particular embodiment, the present invention provides a method ofmodulating the expression of a polypeptide by a cell, comprisingproviding to a cell a lipid particle that consists of or consistsessentially of a lipid chosen from Table 1 or Table 2, DSPC, Chol andPEG-DMG or PEG-DMA, e.g., in a molar ratio of about 20-60% cationiclipid: 5-25% DSPC:25-55% Chol:0.5-15% PEG-DMG or PEG-DMA, wherein thelipid particle is associated with a nucleic acid capable of modulatingthe expression of the polypeptide. In particular embodiments, the molarlipid ratio is approximately 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In particular embodiments, the therapeutic agent is selected from ansiRNA, a microRNA, an antisense oligonucleotide, and a plasmid capableof expressing an siRNA, a microRNA, or an antisense oligonucleotide, andwherein the siRNA, microRNA, or antisense RNA comprises a polynucleotidethat specifically binds to a polynucleotide that encodes thepolypeptide, or a complement thereof, such that the expression of thepolypeptide is reduced.

In other embodiments, the nucleic acid is a plasmid that encodes thepolypeptide or a functional variant or fragment thereof, such thatexpression of the polypeptide or the functional variant or fragmentthereof is increased.

In related embodiments, the present invention provides a method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is selected from an siRNA, a microRNA, an antisenseoligonucleotide, and a plasmid capable of expressing an siRNA, amicroRNA, or an antisense oligonucleotide, and wherein the siRNA,microRNA, or antisense RNA comprises a polynucleotide that specificallybinds to a polynucleotide that encodes the polypeptide, or a complementthereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molarratio of about 20-60% cationic lipid: 5-25% DSPC:25-55% Chol:0.5-15%PEG-DMG or PEG-DMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In another related embodiment, the present invention includes a methodof treating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject apharmaceutical composition of the present invention, wherein thetherapeutic agent is a plasmid that encodes the polypeptide or afunctional variant or fragment thereof.

In one embodiment, the pharmaceutical composition comprises a lipidparticle that consists of or consists essentially of a lipid chosen fromTable 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g., in a molarratio of about 20-60% cationic lipid: 5-25% DSPC:25-55% Chol:0.5-15%PEG-DMG or PEG-DMA, wherein the lipid particle is associated with thetherapeutic nucleic acid. In particular embodiments, the molar lipidratio is approximately 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

The present invention further provides a method of inducing an immuneresponse in a subject, comprising providing to the subject thepharmaceutical composition of the present invention, wherein thetherapeutic agent is an immunostimulatory oligonucleotide. In certainembodiments, the immune response is a humoral or mucosal immuneresponse. In one embodiment, the pharmaceutical composition comprises alipid particle that consists of or consists essentially of a lipidchosen from Table 1 or Table 2, DSPC, Chol and PEG-DMG or PEG-DMA, e.g.,in a molar ratio of about 20-60% cationic lipid: 5-25% DSPC:25-55%Chol:0.5-15% PEG-DMG or PEG-DMA, wherein the lipid particle isassociated with the therapeutic nucleic acid. In particular embodiments,the molar lipid ratio is approximately 40/10/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA), 35/15/40/10 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA) or 52/13/30/5 (mol % cationiclipid/DSPC/Chol/PEG-DMG or PEG-DMA). In another group of embodiments,the neutral lipid in these compositions is replaced with POPC, DPPC,DOPE or SM.

In further embodiments, the pharmaceutical composition is provided tothe subject in combination with a vaccine or antigen. Thus, the presentinvention itself provides vaccines comprising a lipid particle of thepresent invention, which comprises an immunostimulatory oligonucleotide,and is also associated with an antigen to which an immune response isdesired. In particular embodiments, the antigen is a tumor antigen or isassociated with an infective agent, such as, e.g., a virus, bacteria, orparasite.

A variety of tumor antigens, infections agent antigens, and antigensassociated with other disease are well known in the art and examples ofthese are described in references cited herein. Examples of antigenssuitable for use in the present invention include, but are not limitedto, polypeptide antigens and DNA antigens. Specific examples of antigensare Hepatitis A, Hepatitis B, small pox, polio, anthrax, influenza,typhus, tetanus, measles, rotavirus, diphtheria, pertussis,tuberculosis, and rubella antigens. In a preferred embodiment, theantigen is a Hepatitis B recombinant antigen. In other aspects, theantigen is a Hepatitis A recombinant antigen. In another aspect, theantigen is a tumor antigen. Examples of such tumor-associated antigensare MUC-1, EBV antigen and antigens associated with Burkitt's lymphoma.In a further aspect, the antigen is a tyrosinase-related protein tumorantigen recombinant antigen. Those of skill in the art will know ofother antigens suitable for use in the present invention.

Tumor-associated antigens suitable for use in the subject inventioninclude both mutated and non-mutated molecules that may be indicative ofsingle tumor type, shared among several types of tumors, and/orexclusively expressed or overexpressed in tumor cells in comparison withnormal cells. In addition to proteins and glycoproteins, tumor-specificpatterns of expression of carbohydrates, gangliosides, glycolipids andmucins have also been documented. Exemplary tumor-associated antigensfor use in the subject cancer vaccines include protein products ofoncogenes, tumor suppressor genes and other genes with mutations orrearrangements unique to tumor cells, reactivated embryonic geneproducts, oncofetal antigens, tissue-specific (but not tumor-specific)differentiation antigens, growth factor receptors, cell surfacecarbohydrate residues, foreign viral proteins and a number of other selfproteins.

Specific embodiments of tumor-associated antigens include, e.g., mutatedantigens such as the protein products of the Ras p21 protooncogenes,tumor suppressor p53 and BCR-abl oncogenes, as well as CDK4, MUM1,Caspase 8, and Beta catenin; overexpressed antigens such as galectin 4,galectin 9, carbonic anhydrase, Aldolase A, PRAME, Her2/neu, ErbB-2 andKSA, oncofetal antigens such as alpha fetoprotein (AFP), human chorionicgonadotropin (hCG); self antigens such as carcinoembryonic antigen (CEA)and melanocyte differentiation antigens such as Mart 1/Melan A, gp100,gp75, Tyrosinase, TRP1 and TRP2; prostate associated antigens such asPSA, PAP, PSMA, PSM-P1 and PSM-P2; reactivated embryonic gene productssuch as MAGE 1, MAGE 3, MAGE 4, GAGE 1, GAGE 2, BAGE, RAGE, and othercancer testis antigens such as NY-ESO1, SSX2 and SCP1; mucins such asMuc-1 and Muc-2; gangliosides such as GM2, GD2 and GD3, neutralglycolipids and glycoproteins such as Lewis (y) and globo-H; andglycoproteins such as Tn, Thompson-Freidenreich antigen (TF) and sTn.Also included as tumor-associated antigens herein are whole cell andtumor cell lysates as well as immunogenic portions thereof, as well asimmunoglobulin idiotypes expressed on monoclonal proliferations of Blymphocytes for use against B cell lymphomas.

Pathogens include, but are not limited to, infectious agents, e.g.,viruses, that infect mammals, and more particularly humans. Examples ofinfectious virus include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such asHIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g.,dengue viruses, encephalitis viruses, yellow fever viruses);Coronoviridae (e.g., coronaviruses); Rhabdoviradae (e.g., vesicularstomatitis viruses, rabies viruses); Coronaviridae (e.g.,coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses,rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae(e.g., parainfluenza viruses, mumps virus, measles virus, respiratorysyncytial virus); Orthomyxoviridae (e.g., influenza viruses);Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses andNairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae(e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae;Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae herpes simplex virus (HSV) 1 and 2,varicella zoster virus, cytomegalovirus (CMV), herpes virus; Poxyiridae(variola viruses, vaccinia viruses, pox viruses); and Iridoviridae(e.g., African swine fever virus); and unclassified viruses (e.g., theetiological agents of Spongiform encephalopathies, the agent of deltahepatitis (thought to be a defective satellite of hepatitis B virus),the agents of non-A, non-B hepatitis (class 1=internally transmitted;class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk andrelated viruses, and astroviruses).

Also, gram negative and gram positive bacteria serve as antigens invertebrate animals. Such gram positive bacteria include, but are notlimited to Pasteurella species, Staphylococci species, and Streptococcusspecies. Gram negative bacteria include, but are not limited to,Escherichia coli, Pseudomonas species, and Salmonella species. Specificexamples of infectious bacteria include but are not limited to:Helicobacter pyloris, Borelia burgdorferi, Legionella pneumophilia,Mycobacteria sps (e.g., M. tuberculosis, M. avium, M. intracellulare, M.kansaii, M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic sps.), Streptococcuspneumoniae, pathogenic Campylobacter sp., Enterococcus sp., Haemophilusinfuenzae, Bacillus antracis, corynebacterium diphtheriae,corynebacterium sp., Erysipelothrix rhusiopathiae, Clostridiumperfringers, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides sp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidium, Treponemapertenue, Leptospira, Rickettsia, and Actinomyces israelli.

Additional examples of pathogens include, but are not limited to,infectious fungi that infect mammals, and more particularly humans.Examples of infectious fingi include, but are not limited to:Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis,Blastomyces dermatitidis, Chlamydia trachomatis, Candida albicans.Examples of infectious parasites include Plasmodium such as Plasmodiumfalciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax.Other infectious organisms (i.e., protists) include Toxoplasma gondii.

In one embodiment, the formulations of the invention can be used tosilence or modulate a target gene such as but not limited to FVII, Eg5,PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGF beta gene, Erb-B gene, Src gene,CRK gene, GRB2 gene, RAS gene, MEKK gene, .INK gene, RAF gene, Erk1/2gene, PCNA(p21) gene, MYB gene, JUN gene, FOS gene, BCL-2 gene, Cyclin Dgene, VEGF gene, EGFR gene, Cyclin A gene, Cyclin E gene, WNT-1 gene,beta-catenin gene, c-MET gene, PKC gene, NFKB gene, STAT3 gene, survivingene, Her2/Neu gene, SORT1 gene, XBP1 gene, topoisomerase I gene,topoisomerase II alpha gene, p73 gene, p21(WAF1/CIP1) gene, p27(KIP1)gene, PPM1D gene, RAS gene, caveolin I gene, MIB I gene, MTAI gene, M68gene, tumor suppressor genes, p53 tumor suppressor gene, p53 familymember DN-p63, pRb tumor suppressor gene, APC1 tumor suppressor gene,BRCA1 tumor suppressor gene, PTEN tumor suppressor gene, mLL fusiongene, BCR/ABL fusion gene, TEL/AML1 fusion gene, EWS/FLI1 fusion gene,TLS/FUS1 fusion gene, PAX3/FKHR fusion gene, AML1/ETO fusion gene, alphav-integrin gene, Flt-1 receptor gene, tubulin gene, Human PapillomaVirus gene, a gene required for Human Papilloma Virus replication, HumanImmunodeficiency Virus gene, a gene required for Human ImmunodeficiencyVirus replication, Hepatitis A Virus gene, a gene required for HepatitisA Virus replication, Hepatitis B Virus gene, a gene required forHepatitis B Virus replication, Hepatitis C Virus gene, a gene requiredfor Hepatitis C Virus replication, Hepatitis D Virus gene, a generequired for Hepatitis D Virus replication, Hepatitis E Virus gene, agene required for Hepatitis E Virus replication, Hepatitis F Virus gene,a gene required for Hepatitis F Virus replication, Hepatitis G Virusgene, a gene required for Hepatitis G Virus replication, Hepatitis HVirus gene, a gene required for Hepatitis H Virus replication,Respiratory Syncytial Virus gene, a gene that is required forRespiratory Syncytial Virus replication, Herpes Simplex Virus gene, agene that is required for Herpes Simplex Virus replication, herpesCytomegalovirus gene, a gene that is required for herpes Cytomegalovirusreplication, herpes Epstein Barr Virus gene, a gene that is required forherpes Epstein Barr Virus replication, Kaposi's Sarcoma-associatedHerpes Virus gene, a gene that is required for Kaposi'sSarcoma-associated Herpes Virus replication, JC Virus gene, human genethat is required for JC Virus replication, myxovirus gene, a gene thatis required for myxovirus gene replication, rhinovirus gene, a gene thatis required for rhinovirus replication, coronavirus gene, a gene that isrequired for coronavirus replication, West Nile Virus gene, a gene thatis required for West Nile Virus replication, St. Louis Encephalitisgene, a gene that is required for St. Louis Encephalitis replication,Tick-borne encephalitis virus gene, a gene that is required forTick-borne encephalitis virus replication, Murray Valley encephalitisvirus gene, a gene that is required for Murray Valley encephalitis virusreplication, dengue virus gene, a gene that is required for dengue virusgene replication, Simian Virus 40 gene, a gene that is required forSimian Virus 40 replication, Human T Cell Lymphotropic Virus gene, agene that is required for Human T Cell Lymphotropic Virus replication,Moloney-Murine Leukemia Virus gene, a gene that is required forMoloney-Murine Leukemia Virus replication, encephalomyocarditis virusgene, a gene that is required for encephalomyocarditis virusreplication, measles virus gene, a gene that is required for measlesvirus replication, Vericella zoster virus gene, a gene that is requiredfor Vericella zoster virus replication, adenovirus gene, a gene that isrequired for adenovirus replication, yellow fever virus gene, a genethat is required for yellow fever virus replication, poliovirus gene, agene that is required for poliovirus replication, poxvirus gene, a genethat is required for poxvirus replication, plasmodium gene, a gene thatis required for plasmodium gene replication, Mycobacterium ulceransgene, a gene that is required for Mycobacterium ulcerans replication,Mycobacterium tuberculosis gene, a gene that is required forMycobacterium tuberculosis replication, Mycobacterium leprae gene, agene that is required for Mycobacterium leprae replication,Staphylococcus aureus gene, a gene that is required for Staphylococcusaureus replication, Streptococcus pneumoniae gene, a gene that isrequired for Streptococcus pneumoniae replication, Streptococcuspyogenes gene, a gene that is required for Streptococcus pyogenesreplication, Chlamydia pneumoniae gene, a gene that is required forChlamydia pneumoniae replication, Mycoplasma pneumoniae gene, a genethat is required for Mycoplasma pneumoniae replication, an integringene, a selectin gene, complement system gene, chemokine gene, chemokinereceptor gene, GCSF gene, Gro1 gene, Gro2 gene, Gro3 gene, PF4 gene, MIGgene, Pro-Platelet Basic Protein gene, MIP-1I gene, MIP-1J gene, RANTESgene, MCP-1 gene, MCP-2 gene, MCP-3 gene, CMBKR1 gene, CMBKR2 gene,CMBKR3 gene, CMBKR5v, AIF-1 gene, I-309 gene, a gene to a component ofan ion channel, a gene to a neurotransmitter receptor, a gene to aneurotransmitter ligand, amyloid-family gene, presenilin gene, HD gene,DRPLA gene, SCA1 gene, SCA2 gene, MJD1 gene, CACNL1A4 gene, SCAT gene,SCA8 gene, allele gene found in LOH cells, or one allele gene of apolymorphic gene.

DEFINITIONS

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, tert-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

The term “acyl” refers to hydrogen, alkyl, partially saturated or fullysaturated cycloalkyl, partially saturated or fully saturatedheterocycle, aryl, and heteroaryl substituted carbonyl groups. Forexample, acyl includes groups such as (C1-C20)alkanoyl (e.g., formyl,acetyl, propionyl, butyryl, valeryl, caproyl, t-butylacetyl, etc.),(C3-C20)cycloalkylcarbonyl (e.g., cyclopropylcarbonyl,cyclobutylcarbonyl, cyclopentylcarbonyl, cyclohexylcarbonyl, etc.),heterocyclic carbonyl (e.g., pyrrolidinylcarbonyl,pyrrolid-2-one-5-carbonyl, piperidinylcarbonyl, piperazinylcarbonyl,tetrahydrofuranylcarbonyl, etc.), aroyl (e.g., benzoyl) and heteroaroyl(e.g., thiophenyl-2-carbonyl, thiophenyl-3-carbonyl, furanyl-2-carbonyl,furanyl-3-carbonyl, 1H-pyrroyl-2-carbonyl, 1H-pyrroyl-3-carbonyl,benzo[b]thiophenyl-2-carbonyl, etc.).

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclichydrocarbon ring system, wherein any ring atom can be substituted.Examples of aryl moieties include, but are not limited to, phenyl,naphthyl, anthracenyl, and pyrenyl.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic,8-12 membered bicyclic, or 11-14 membered tricyclic ring system having1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9heteroatoms if tricyclic, said heteroatoms selected from O, N, or S(e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S ifmonocyclic, bicyclic, or tricyclic, respectively), wherein any ring atomcan be substituted. The heteroaryl groups herein described may alsocontain fused rings that share a common carbon-carbon bond. The term“alkylheterocyle” refers to a heteroaryl wherein at least one of thering atoms is substituted with alkyl, alkenyl or alkynyl

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with the radical of a specifiedsubstituent including, but not limited to: halo, alkyl, alkenyl,alkynyl, aryl, heterocyclyl, thiol, alkylthio, oxo, thioxy, arylthio,alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl,arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl,alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl,haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino,alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl,carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl,aralkoxycarbonyl, carboxylic acid, sulfonic acid, sulfonyl, phosphonicacid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understoodthat the substituent may be further substituted. Exemplary substituentsinclude amino, alkylamino, dialkylamino, and cyclic amino compounds.

“Halogen” means fluoro, chloro, bromo and iodo.

The terms “alkylamine” and “dialkylamine” refer to —NH(alkyl) and —N(alkyl)₂ radicals respectively.

The term “alkylphosphate” refers to —O—P(Q′)(Q″)-O—R, wherein Q′ and Q″are each independently O, S, N(R)₂, optionally substituted alkyl oralkoxy; and R is optionally substituted alkyl, ω-aminoalkyl orω-(substituted)aminoalkyl.

The term “alkylphosphorothioate” refers to an alkylphosphate wherein atleast one of Q′ or Q″ is S.

The term “alkylphosphonate” refers to an alkylphosphate wherein at leastone of Q′ or Q″ is alkyl.

The terem “hydroxyalkyl” means —O-alkyl radical.

The term “alkylheterocycle” refers to an alkyl where at least onemethylene has been replaced by a heterocycle.

The term “ω-aminoalkyl” refers to -alkyl-NH₂ radical. And the term“ω-(substituted)aminoalkyl refers to an ω-aminoalkyl wherein at leastone of the H on N has been replaced with alkyl.

The term “ω-phosphoalkyl” refers to -alkyl-O—P(Q′)(Q″)—O—R, wherein Q′and Q″ are each independently O or S and R optionally substituted alkyl.

The term “ω-thiophosphoalkyl refers to ω-phosphoalkyl wherein at leastone of Q′ or Q″ is S.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et. al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

The compounds of the present invention may be prepared by known organicsynthesis techniques, including the methods described in more detail inthe Examples.

EXAMPLES Example 1 Synthesis of methanesulfonic acidoctadeca-9,12-dienyl ester 2

To a solution of the alcohol 1 (26.6 g, 100 mmol) in dichloromethane(100 mL), triethylamine (13.13 g, 130 mmol) was added and this solutionwas cooled in an ice-bath. To this cold solution, a solution of mesylchloride (12.6 g, 110 mmol) in dichloromethane (60 mL) was addeddropwise and after the completion of the addition, the reaction mixturewas allowed to warm to ambient temperature and stirred overnight. TheTLC of the reaction mixture showed the completion of the reaction. Thereaction mixture was diluted with dichloromethane (200 mL), washed withwater (200 mL), satd. NaHCO₃ (200 mL), brine (100 mL) and dried (NaSO₄).The organic layer was concentrated to get the crude product which waspurified by column chromatography (silica gel) using 0-10% Et₂O inhexanes. The pure product fractions were combined and concentrated toobtain the pure product 2 as colorless oil (30.6 g, 89%). ¹H NMR (CDCl₃,400 MHz) δ=5.42-5.21 (m, 4H), 4.20 (t, 2H), 3.06 (s, 3H), 2.79 (t, 2H),2.19-2.00 (m, 4H), 1.90-1.70 (m, 2H), 1.06-1.18 (m, 18H), 0.88 (t, 3H).¹³C NMR (CDCl₃) δ=130.76, 130.54, 128.6, 128.4, 70.67, 37.9, 32.05,30.12, 29.87, 29.85, 29.68, 29.65, 29.53, 27.72, 27.71, 26.15, 25.94,23.09, 14.60. MS. Molecular weight calculated for C₁₉H₃₆O₃S, Cal.344.53; Found 343.52 (M−H⁻).

Synthesis of 18-Bromo-octadeca-6,9-diene 3

The mesylate 2 (13.44 g, 39 mmol) was dissolved in anhydrous ether (500mL) and to it the MgBr.Et₂O complex (30.7 g, 118 mmol) was added underargon and the mixture was refluxed under argon for 26 h after which theTLC showed the completion of the reaction. The reaction mixture wasdiluted with ether (200 mL) and ice-cold water (200 mL) was added tothis mixture and the layers were separated. The organic layer was washedwith 1% aqueous K₂CO₃ (100 mL), brine (100 mL) and dried (Anhyd.Na₂SO₄). Concentration of the organic layer provided the crude productwhich was further purified by column chromatography (silica gel) using0-1% Et₂O in hexanes to isolate the bromide 3 (12.6 g, 94%) as acolorless oil. ¹H NMR (CDCl₃, 400 MHz) δ=5.41-5.29 (m, 4H), 4.20 (d,2H), 3.40 (t, J=7 Hz, 2H), 2.77 (t, J=6.6 Hz, 2H), 2.09-2.02 (m, 4H),1.88-1.00 (m, 2H), 1.46-1.27 (m, 18H), 0.88 (t, J=3.9 Hz, 3H). ¹³C NMR(CDCl₃) δ=130.41, 130.25, 128.26, 128.12, 34.17, 33.05, 31.75, 29.82,29.57, 29.54, 29.39, 28.95, 28.38, 27.42, 27.40, 25.84, 22.79, 14.28.

Synthesis of 18-Cyano-octadeca-6,9-diene 4

To a solution of the mesylate (3.44 g, 10 mmol) in ethanol (90 mL), asolution of KCN (1.32 g, 20 mmol) in water (10 mL) was added and themixture was refluxed for 30 min after which, the TLC of the reactionmixture showed the completion of the reaction after which, ether (200mL) was added to the reaction mixture followed by the addition of water.The reaction mixture was extracted with ether and the combined organiclayers was washed with water (100 mL), brine (200 mL) and dried.Concentration of the organic layer provided the crude product which waspurified by column chromatography (0-10% Et₂O in hexanes). The pureproduct 4 was isolated as colorless oil (2 g, 74%). ¹H NMR (CDCl₃, 400MHz) δ=5.33-5.22 (m, 4H), 2.70 (t, 2H), 2.27-2.23 (m, 2H), 2.00-1.95 (m,4H), 1.61-1.54 (m, 2H), 1.39-1.20 (m, 18H), 0.82 (t, 3H). ¹³C NMR(CDCl₃) δ=130.20, 129.96, 128.08, 127.87, 119.78, 70.76, 66.02, 32.52,29.82, 29.57, 29.33, 29.24, 29.19, 29.12, 28.73, 28.65, 27.20, 27.16,25.62, 25.37, 22.56, 17.10, 14.06. MS. Molecular weight calculated forC₁₉H₃₃N, Cal. 275.47; Found 276.6 (M−H⁻).

Synthesis of Heptatriaconta-6,9,28,31-tetraen-19-one 7

To a flame dried 500 mL 2NRB flask, freshly activated Mg turnings (0.144g, 6 mmol) were added and the flask was equipped with a magnetic stirbar and a reflux condenser. This set-up was degassed, flushed with argonand 10 mL of anhydrous ether was added to the flask via syringe. Thebromide 3 (1.65 g, 5 mmol) was dissolved in anhydrous ether (10 mL) andadded dropwise via syringe to the flask. An exothermic reaction wasnoticed (to confirm/accelerate the Grignard reagent formation, 2 mg ofiodine was added and immediate decolorization was observed confirmingthe formation of the Grignard reagent) and the ether started refluxing.After the completion of the addition the reaction mixture was kept at35° C. for 1 h and then cooled in ice bath. The cyanide 4 (1.38 g, 5mmol) was dissolved in anhydrous ether (20 mL) and added dropwise to thereaction mixture with stirring. An exothermic reaction was observed andthe reaction mixture was stirred overnight at ambient temperature. Thereaction was quenched by adding 10 mL of acetone dropwise followed byice cold water (60 mL). The reaction mixture was treated with aq. H₂SO₄(10% by volume, 200 mL) until the solution became homogeneous and thelayers were separated. The aq. phase was extracted with ether (2×100mL). The combined ether layers were dried (Na₂SO₄) and concentrated toget the crude product which was purified by column (silica gel, 0-10%ether in hexanes) chromatography. The pure product fractions wereevaporated to provide the pure ketone 7 as a colorless oil (2 g, 74%).¹H NMR (CDCl₃, 400 MHz) δ=5.33-5.21 (m, 8H), 2.69 (t, 4H), 2.30 (t, 4H),2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m, 18H), 0.82 (t, 3H).¹³C NMR (CDCl₃) δ=211.90, 130.63, 130.54, 128.47, 128.41, 43.27, 33.04,32.01, 30.93, 29.89, 29.86, 29.75, 29.74, 27.69, 26.11, 24.35, 23.06,14.05. MS. Molecular weight calculated for C₃₇H₆₆O, Cal. 526.92; Found528.02 (M+H⁺).

Example 2 Alternative Synthesis of the Ketone 7

Synthesis of Compound 6b

To a flame dried 500 mL RB flask, freshly activated Mg turnings (2.4 g,100 mmol) were added and the flask was equipped with a magnetic stirbar, an addition funnel and a reflux condenser. This set-up was degassedand flushed with argon and 10 mL of anhydrous ether was added to theflask via syringe. The bromide 3 (26.5 g, 80.47 mmol) was dissolved inanhydrous ether (50 mL) and added to the addition funnel. About 5 mL ofthis ether solution was added to the Mg turnings while stirringvigorously. An exothermic reaction was noticed (to confirm/acceleratethe Grignard reagent formation, 5 mg of iodine was added and immediatedecolorization was observed confirming the formation of the Grignardreagent) and the ether started refluxing. The rest of the solution ofthe bromide was added dropwise while keeping the reaction under gentlereflux by cooling the flask in water. After the completion of theaddition the reaction mixture was kept at 35° C. for 1 h and then cooledin ice bath. Ethyl formate (2.68 g, 36.2 mmol) was dissolved inanhydrous ether (40 mL) and transferred to the addition funnel and addeddropwise to the reaction mixture with stirring. An exothermic reactionwas observed and the reaction mixture started refluxing. After theinitiation of the reaction the rest of the ethereal solution of formatewas quickly added as a stream and the reaction mixture was stirred for afurther period of 1 h at ambient temperature. The reaction was quenchedby adding 10 mL of acetone dropwise followed by ice cold water (60 mL).The reaction mixture was treated with aq. H₂SO₄ (10% by volume, 300 mL)until the solution became homogeneous and the layers were separated. Theaq. phase was extracted with ether (2×100 mL). The combined ether layerswere dried (Na₂SO₄) and concentrated to get the crude product which waspurified by column (silica gel, 0-10% ether in hexanes) chromatography.The slightly less polar fractions were concentrated to get the formate6a (1.9 g) and the pure product fractions were evaporated to provide thepure product 6b as a colorless oil (14.6 g, 78%).

Synthesis of Compound 7

To a solution of the alcohol 6b (3 g, 5.68 mmol) in CH₂Cl₂ (60 mL),freshly activated 4A molecular sieves (50 g) were added and to thissolution powdered PCC (4.9 g, 22.7 mmol) was added portion wise over aperiod of 20 minutes and the mixture was further stirred for 1 hour(Note: careful monitoring of the reaction is necessary in order to getgood yields since prolonged reaction times leads to lower yields) andthe TLC of the reaction mixture was followed every 10 minutes (5% etherin hexanes) After completion of the reaction, the reaction mixture wasfiltered through a pad of silica gel and the residue was washed withCH₂Cl₂ (400 mL). The filtrate was concentrated and the thus obtainedcrude product was further purified by column chromatography (silica gel,1% Et₂O in hexanes) to isolate the pure product 7 (2.9 g, 97%) as acolorless oil. ¹H NMR (CDCl₃, 400 MHz) δ=5.33-5.21 (m, 8H), 2.69 (t,4H), 2.30 (t, 4H), 2.05-1.95 (m, 8H), 1.55-1.45 (m, 2H), 1.35-1.15 (m,18H), 0.82 (t, 3H). ¹³C NMR (CDCl₃) δ=211.90, 130.63, 130.54, 128.47,128.41, 43.27, 33.04, 32.01, 30.93, 29.89, 29.86, 29.75, 29.74, 27.69,26.11, 24.35, 23.06, 14.05. MS. Molecular weight calculated for C₃₇H₆₆O,Cal. 526.92; Found 528.02 (M+H⁺).

Example 3 Synthesis of Unsymmetric Ketones 25 and 27

Synthesis of heptatriaconta-6,9,28-trien-19-one 25

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (132 mg,0.0054 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide 24 (1.8 g, 0.0054 mol) was dissolved in anhydrousether (10 mL) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide 4 (0.5 g, 0.0018 mol) was dissolved in dry THF (5 mL) andadded dropwise to the reaction with stirring. An exothermic reaction wasobserved and the reaction mixture was refluxed (at 70° C.) for 12 h andquenched with ammonium chloride solution. It was then treated with 25%HCl solution until the solution became homogenous and the layers wereseparated. The aqueous phase was extracted with ether. The combinedether layers were dried and concentrated to get the crude product whichwas purified by column chromatography. The pure product fractions wereevaporated to provide the pure ketone 25 as colorless oil.

Yield: 0.230 g (24%). ¹H-NMR (CDCl3, 400 MHz): δ=5.37-5.30 (m, 6H),2.77-2.74 (t, 2H), 2.38-2.34 (t, 4H), 2.05-1.95 (m, 8H), 1.56-1.52 (m,4H), 1.35-1.25 (m, aliphatic protons), 0.89-0.85 (t, 6H). IR(cm−1):2924, 2854,1717,1465,1049,721.

Synthesis of heptatriaconta-6,9-dien-19-one 27

To a flame dried 500 mL 2NRB flask, a freshly activated Mg turnings(0.144 g, 6 mmol) is added and the flask is equipped with a magneticstir bar and a reflux condenser. This set-up is degassed and flushedwith argon and 10 mL of anhydrous ether is added to the flask viasyringe. The commercially available bromide 26 (2.65 g, 5 mmol) isdissolved in anhydrous ether (10 mL) and added dropwise via syringe tothe flask. After the completion of the addition the reaction mixture iskept at 35° C. for 1 h and then cooled in ice bath. The cyanide 4 (1.38g, 5 mmol) is dissolved in anhydrous ether (20 mL) and added dropwise tothe reaction mixture with stirring. An exothermic reaction is observedand the reaction mixture is stirred overnight at ambient temperature.The reaction is quenched by adding 10 mL of acetone dropwise followed byice cold water (60 mL). The reaction mixture is treated with aq. H₂SO₄(10% by volume, 200 mL) until the solution becomes homogeneous and thelayers are separated. The aq. phase is extracted with ether (2×100 mL).The combined ether layers are dried (Na₂SO₄) and concentrated to get thecrude product which is purified by column chromatography to provide thepure ketone 27 as a colorless oil. 1H-NMR (CDCl3, 400 MHz): δ=5.42-5.30(m, 4H), 2.79-2.78 (t, 2H), 2.40-2.37 (t, 4H), 2.08-2.03 (m, 4H),1.58-1.54 (m, 4H), 1.36-1.26 (br m, aliphatic protons), 0.91-0.87 (t,6H). IR (cm−1):2924, 2854, 1716, 1465, 1375, 721.

Example 4 Synthesis of Unsymmetrical Ketones with C₁₂ Chain

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (175 mg,0.0072 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide 28 (1.5 g, 0.006 mol) was dissolved in anhydrousether (7 ml) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide 4 (1 g, 0.0036 mol) was dissolved in anhydrous ether (7mL) and added dropwise to the reaction with stirring. An exothermicreaction was observed and the reaction mixture was refluxed for 12 h andquenched with ammonium chloride solution. It was then treated with 25%HCl solution until the solution becomes homogenous and the layers wereseparated. The aq phase was extracted with ether. The combined etherlayers were dried and concentrated to get the crude product which waspurified by column chromatography. The pure product fractions wereevaporated to provide the pure ketone 29 as colorless oil. Yield: 0.65 g(26%). ¹H-NMR (δ ppm): 5.388-5.302 (m, 4H), 2.77-2.74 (t, 2H), 2.38-2.34(t, 4H), 2.04-2.01 (m, 4H), 1.34-1.18 (m, 36H), 0.89-0.85 (m 6H). IR(cm⁻¹): 3009, 2920, 2851, 1711 (C═O), 1466, 1376, 1261.

Example 5 Synthesis of Unsymmetrical Ketones with C₁₀ Chain 31

To a dry 50 ml 2NRB flask, a freshly activated Mg turnings (266 mg,0.0109 mol) was added and the flask was equipped with a magnetic stirbar and a reflux condenser. This setup was degassed and flushed withnitrogen and 10 mL of anhydrous ether was added to the flask viasyringe. The bromide (2.43 g, 0.0109 mol) was dissolved in anhydrousether (7 ml) and added dropwise via syringe to the flask. An exothermicreaction was noticed (reaction initiated with dibromoethane) and theether started refluxing. After completion of the addition the reactionmixture was kept at 35° C. for 1 h and then cooled in ice bath to 10-15°C. The cyanide (1 g, 0.0036 mol) was dissolved in anhydrous ether (7 mL)and added dropwise to the reaction with stirring. An exothermic reactionwas observed and the reaction mixture was stirred at ambient temperaturefor 2 hr. THF (4 ml) was added to the reaction mixture and it was warmedto 45-50° C. for 4 hr till the cyano derivative was complete consumed.The reaction was quenched by adding 3 mL of acetone dropwise followed byice cold water. The reaction mixture was treated with 25% HCl solutionuntil the solution becomes homogenous and the layers were separated. Theaq. phase was extracted with ether. The combined ether layers were driedand concentrated to get the crude product which was purified by columnchromatography. The pure product fractions were evaporated to providethe pure ketone as colorless oil. Yield: 0.93 gms (61%). ¹H-NMR (δ ppm):5.37-5.302 (m, 4H), 2.77-2.74 (t, 2H), 2.38-2.34 (t, 4H), 2.05-2.00 (m,4H), 1.55-1.52 (m, 2H), 1.35-1.24 (m, 34H), 0.89-0.84 (m 6H). IR (cm⁻¹):3009, 2925, 2854, 1717 (C═O), 1465, 1376.

Example 6 Synthesis of Unsymmetrical Ketones with Cholesterol 33

Using a similar procedure to that used for the synthesis of ketone 31,the cholesteryl chloride on conversion to the corresponding magnesiumchloride followed by addition to the linoleyl cyanide provided theketone 33.

Example 7 Synthesis of Unsymmetrical Ketones with Cholesterol 35

The treatment of the cholesterolchloroformate with 3-bromopropylamineprovided the bromide 34 which is converted to the corresponding Grignardreagent 34a which on treatment with the linoleyl cyanide provided thecorresponding unsymmetrical ketone 35 in good yield.

Example 8 Synthesis of Unsymmetric Ketone 40

Synthesis of Compound 37

To a 500 ml two neck RBF containing LiAlH₄ (1.02 g, 0.0269 mol) wasadded anhydrous THF (20 mL) at room temperature under nitrogenatmosphere. The suspension was stirred for 1 h at room temperature andthen cooled to 0° C. To this mixture was added a solution of compound 1(5 g, 0.01798 mol) in anhydrous THF (50 mL) slowly maintaining theinside temperature 0° C. After completion of the addition, reactionmixture was warmed to ambient temperature and stirred for 1 h. Progressof the reaction was monitored by TLC. Upon completion of the reaction,mixture was cooled to 0° C. and quenched with sat. solution of aq.Na₂SO₄. Reaction mixture was stirred for 30 minutes and solid formed wasfiltered through celite bed and washed with ethyl acetate (100 mL).Filtrate and washings were combined and evaporated on rotary evaporatorto afford the compound 37 as colorless liquid, which was taken as suchfor the next stage without any purification. Yield: (4.5 g, 95%); ¹H NMR(400 MHz, CDCl₃) δ=5.39-5.28 (m, 6H), 3.64-3.61 (t, 2H), 2.81-2.78 (t,4H), 2.10-2.01 (m, 4H), 1.59-1.51 (m, 2H), 1.29-1.22 (m, aliphaticprotons), 0.98-0.94 (t, 3H).

Synthesis of Compound 38

Compound 37 (14 g, 0.0530 mol) was dissolved in DCM (300 ml) in a 500 mltwo neck RBF and cooled to 0° C. To this solution was addedtriethylamine (29.5 ml, 0.2121 mol) slowly under inert atmosphere.Reaction mixture was then stirred for 10-15 minutes and to it mesylchloride (6.17 mL, 0.0795 mol)) was added slowly. After completeaddition, the reaction mixture was allowed to warm to ambienttemperature and stirred for 20 h. Reaction was monitored by TLC. Uponcompletion, the reaction mixture was diluted with water (200 mL) stirredfor few minutes and organic layer was separated. Organic phase wasfurther washed with brine (1×70 mL), dried over Na₂SO₄ and solvent wasremoved on rotary evaporator to get the crude compound 38 as brown oilwhich was used as such for next reaction. Yield: (17 g, 93%) ¹H NMR (400MHz, CDCl₃) δ=5.39-5.31 (m, 6H), 4.22-4.19 (t, 2H), 2.99 (s, 3H),2.81-2.78 (m, 4H), 2.08-2.01 (m, 4H), 1.75. 1.69 (m, 2H), 1.39-1.29 (m,aliphatic protons), 0.98-0.94 (t, 3H).

Synthesis of Compound 39

The mesylate 38 (10 g, 0.2923 mol) was dissolved in (300 mL) anhydrousether in a 1000 mL two neck RBF and MgBr₂.Et₂O complex (22.63 g, 0.0877mol) was added into it under nitrogen atmosphere. Resulting mixture wasthen heated to reflux for 26 h. After completion of the reaction (byTLC), reaction mixture was diluted with ether (300 mL) and ice coldwater (200 mL) and ether layer was separated out. Organic layer was thenwashed with 1% aq. K₂CO₃ (100 mL) followed by brine (80 mL). Organicphase was then dried over anhydrous Na₂SO₄ and solvent was evaporatedoff under vacuum to give the crude material which was chromatographed onsilca gel (60-120 mesh) using 0-1% ethyl acetate in hexane as elutingsystem to yield the desired compound 39 as oil. Yield: (7 g, 73%) ¹H NMR(400 MHz, CDCl₃) δ=5.39-5.31 (m, 6H), 3.41-3.37 (t, 2H), 2.81-2.78 (m,4H), 2.08-2.02 (m, 4H), 1.86-1.80 (m, 2H), 1.42-1.29 (m, aliphaticprotons), 0.98-0.94 (t, 3H).

Synthesis of Unsymetric Ketone 40

To a flame dried 500 mL two neck RBF, equipped with magnetic stir barand a reflux condenser, freshly activated Mg turnings (0.88 g, 0.03636mol) were added. This set up was degassed, flushed with argon and ether(150 mL) was added into it. Few drops of bromo compound 4 (11.89 g,0.03636 mol) in 50 mL ether was added at the beginning to initiate thereaction (note: catalytic amount of 1,2-dibromo ethane was also added toaccelerate formation of grignard reagent). Upon initiation, theremaining solution of bromo compound was added slowly to the refluxingethereal solution. After complete addition, the reaction mixture wasrefluxed at 40° C. for 1.5 h. It was then cooled to 10° C. and thelinoleyl cyanide 4 (5 g, 0.01818 mol) in 30 mL of dry ether was addeddrop wise and the resulting mixture was then heated to reflux for 20 hat 40° C. Progress of the reaction was monitored by TLC. After completeconsumption of the cyano derivative 40 (by TLC), mixture was cooled toroom temperature and quenched with 30 mL of acetone followed by (50 mL)ice water. This solution was further acidified with 10% HCl solution andether layer was separated out. Aqueous phase was further extracted withdiethyl ether (2×100 mL). Removal of the solvent after drying overanhydrous Na₂SO₄ afforded the crude ketone which was purified by silicagel column chromatography (100-200 mesh) using 0-5% ether in hexane aseluting system to give the title compound 40 as pale yellow oil. Yield:(4.8 g, 50.5%) ¹H NMR (400 MHz, CDCl₃) δ=5.38-5.28 (m, 10H), 2.80-2.74(m, 6H), 2.38-2.34 (t, 4H), 2.08-2.00 (m, 8H), 1.55-1.52 (m, 4H),1.35-1.26 (m, aliphatic protons), 0.98-0.94 (t, 3H), 0.89-0.85 (t, 3H).HPLC-98.04%.

Example 9 Synthesis of Ketal 506

Dihydroxylation of Compound 501

A cooled (0° C.) solution of OsO₄ (0.01 eq) and NMO (1 eq) in water (100mL) was treated dropwise with a solution of compound 501 (10.3 g, 61mmol) in acetone (100 mL). After 5 h at r.t., the solvent was removedand the aqueous residue was partitioned with EtOAc. After aqueousworkup, column chromatography gave compound 502 (9.2 g, 74%) as acolorless solid. ¹H NMR (DMSO-d₆, 400 MHz) δ=4.86 (t, J=4.0 Hz, 2H),3.96 (m, 2H), 3.36-3.26 (m, 2H), 3.05 (dd, J=9.8 and 3.8 Hz, 2H), 1.38(s, 9H); Electrospray MS (+ve): Molecular weight for C9H18NO4 (M+H)⁺Calc. 204.0; Found 104.0 (M+H minus Boc).

Preparation of Compound 503

A DCM solution (20 mL) of compound 502 (2.5 g, 12.3 mmol) was treatedwith HCl in dioxane (4 M, 80 mL) over 18 h. Et₂O (200 mL) was added andthe resulting brown solid (compound 503) was collected by filtration(1.73 g, 100%). ¹H NMR (DMSO-d₆, 400 MHz) δ=9.35 (br, 1H), 9.25 (br,1H), 5.6-5.1 (br, 2H), 4.05 (t, J=4.1 Hz, 2H), 3.23-3.13 (m, 2H),2.97-2.89 (m, 2H); Electrospray MS (+ve): Molecular weight for C4H10NO2(M+H)⁺ Calc. 104.0; Found 104.0.

Preparation of Compound 504

A DCM solution (20 mL) of NEt₃ (3 eq) and compound 503 (1.73 g, 12.3mmol) was treated with Cbz-OSu (1.1 eq) over 18 h. Aqueous workup thencolumn chromatography gave pure compound 7 (2.0 g, 68%). ¹H NMR (CDCl₃,400 MHz) δ=7.40-7.26 (m, 5H), 5.04 (s, 2H), 5.92 (d, J=4.5 Hz, 2H), 3.99(m, 2H), 3.44 (dd, J=10.5 and 5.4 Hz, 1H), 3.37 (dd, J=10.9 and 5.3 Hz,1H), 3.14 (dt, J=10.4 and 4.6 Hz, 2H).

Preparation of Compound 505

A mixture of compound 504 (1.85 g, 7.8 mmol), ketone 7 (2.74 g, 5.2mmol) and p-TSA (0.1 eq) was heated under toluene reflux with Dean-Starkapparatus for 3 h. Removal of solvent then column chromatography gavecompound 505 (3.6 g, 93%) as a colorless oil. ¹H NMR (CDCl₃, 400 MHz)δ=7.40-7.26 (m, 5H), 5.43-5.29 (m, 8H), 5.13 (d, J=9.2 Hz, 2H), 4.71 (d,J=4.1 Hz, 2H), 3.88 (dd, J=22.4 and 12.9 Hz, 2H), 3.31 (d, J=12.9 Hz,2H), 2.77 (t, J=6.4 Hz, 4H), 2.09-2.00 (m, 8H), 1.62 (m, 2H), 1.51 (br,2H), 1.40-1.21 (m, 36H), 0.89 (t, J=6.8 Hz, 6H); Electrospray MS (+ve):Molecular weight for C49H80NO4 (M+H)⁺ Calc. 746.6; Found 746.4.

Preparation of Compound 506 (ALNY-104)

A solution of compound 505 (2 g, 2.68 mmol) in hexane (20 mL) was addedin a dropwise fashion to an ice-cold solution of LAH in THF (1 M, 5.4mL). After complete addition, the mixture was heated at 40° C. over 0.5h then cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na₂SO₄ then filtered through celite and reducedto an oil. Column chromatography gave pure 506, 1.32 g, 79% as acolorless oil. ¹H NMR (CDCl₃, 400 MHz) δ=5.43-5.29 (m, 8H), 4.61 (s,2H), 2.97 (d, J=10.9 Hz, 2H), 2.77 (t, J=6.4 Hz, 4H), 2.28 (s, 3H),2.12-2.01 (m, 10H), 1.72 (m, 2H), 1.52 (br, 2H), 1.44-1.23 (m, 36H),0.89 (t, J=6.8 Hz, 6H); ¹³C NMR δ=130.2 (×3), 128.0, 127.9, 115.1, 80.2,82.3, 41.9, 36.8, 35.9, 31.5, 30.0, 29.9, 29.7 (×2), 29.6, 29.5, 29.4,29.3, 27.3, (×2), 27.2, 25.7, 24.3, 23.9, 22.6, 14.0; Electrospray MS(+ve): Molecular weight for C42H76NO2 (M+H)⁺ Calc. 626.6; Found 626.6.

Example 10 Synthesis of Ketal 512

Preparation of Compound 508

Using a procedure analogous to that described for the synthesis ofcompound 502, compound 508 (10 g, 73%) was obtained as a colorless oil.¹H NMR (400 MHz, DMSO) δ 4.65-4.55 (br, 1H), 4.50 (d, J=4.1, 1H), 3.64(m, 1H), 3.49-3.37 (m, 1H), 3.31-3.04 (m, 4H), 1.60 (m, 1H), 1.49-1.27(m, 10H); Electrospray MS (+ve): Molecular weight for C₁₀H₂₀NO₄ (M+H)⁺Calc. 218.1; Found 118.0 (M+H minus Boc).

Preparation of Compound 509

Using a procedure analogous to that described for the synthesis ofcompound 503, compound 509 (7.33 g, 100%) was obtained as a colorlessoil. ¹H NMR (400 MHz, DMSO) δ 9.05 (s, 1H), 8.51 (s, 1H), 5.5-4.7 (br,2H), 3.83-3.73 (m, 1H), 3.73-3.63 (m, 1H), 3.06-2.79 (m, 4H), 1.80 (m,1H), 1.67 (m, 1H); Electrospray MS (+ve): Molecular weight for C₅H₁₂NO₂(M+H)⁺ Calc. 118.1; Found 118.0.

Preparation of Compound 510

Using a procedure analogous to that described for the synthesis ofcompound 504, compound 510 (2.68 g, 82%) was obtained as a colorlessoil. ¹H NMR (400 MHz, DMSO) δ 7.45-7.21 (m, 5H), 5.05 (s, 2H), 4.68 (s,1H), 4.57 (s, 1H), 3.67 (d, J=3.0, 1H), 3.55-3.11 (m, 5H), 1.65 (m, 1H),1.48 (m, 1H); Electrospray MS (+ve): Molecular weight for C₁₃H₁₈NO₄(M+H)⁺ Calc. 252.1; Found 252.0.

Preparation of Compound 511

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 511 (4.7 g, 87%) was obtained as a colorless oil.¹H NMR (400 MHz, CDCl₃) δ 7.45-7.28 (m, 5H), 5.42-5.28 (m, 8H),5.23-5.00 (m, 2H), 4.43-4.33 (m, 1H), 4.27 (d, J=26.5, 1H), 3.94-3.72(m, 1H), 3.61-3.24 (m, 4H), 2.77 (t, J=6.3, 4H), 2.15-1.91 (m, 8H),1.89-1.76 (br, 1H), 1.71-1.60 (m, 2H), 1.54 (m, 2H), 1.42-1.20 (m, 36H),0.88 (t, J=6.7, 6H); Electrospray MS (+ve): Molecular weight forC₅₀H₈₂NO₄ (M+H)⁺ Calc. 760.6; Found 760.4.

Preparation of Compound 512 (ALNY-105)

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 512 (ALNY-105) (0.90 g, 69%) was obtained as acolorless oil. ¹H NMR (400 MHz, CDCl₃) δ 5.49-5.23 (m, 8H), 4.16 (t,J=5.2, 2H), 2.77 (t, J=6.3, 4H), 2.70 (dd, J=11.6, 5.0, 1H), 2.44-2.33(m, 1H), 2.31-2.14 (m, 5H), 2.04 (dd, J=13.6, 6.8, 8H), 1.99-1.88 (m,1H), 1.78-1.63 (m, 2H), 1.56 (m, 2H), 1.48-1.15 (m, 37H), 0.88 (t,J=6.7, 6H). ¹³C NMR=130.2, 130.1 (×2), 127.9 (×2), 72.1, 70.6, 68.1,51.1, 46.3, 38.5, 36.5, 31.5, 30.0, 29.7, 29.6, 29.5 (×2), 29.3 (×3),27.7, 27.2 (×2), 25.6, 24.4, 22.6, 14.1; Electrospray MS (+ve):Molecular weight for C₄₃H₇₈NO₂ (M+H)⁺ Calc. 640.6; Found 640.6.

Example 11 Synthesis of Ketal 519a-g [ALNY-100, 101, 107, 108, 109, 110and 189]

Synthesis of 515

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1 L), was added a solution of 514 (10g, 0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogenatmosphere. After complete addition, reaction mixture was warmed to roomtemperature and then heated to reflux for 4 h. Progress of the reactionwas monitored by TLC. After completion of reaction (by TLC) the mixturewas cooled to 0° C. and quenched with careful addition of saturatedNa₂SO₄ solution. Reaction mixture was stirred for 4 h at roomtemperature and filtered off. Residue was washed well with THF. Thefiltrate and washings were mixed and diluted with 400 mL dioxane and 26mL conc. HCl and stirred for 20 minutes at room temperature. Thevolatilities were stripped off under vacuum to furnish the hydrochloridesalt of 515 as a white solid. Yield: 7.12 g ¹H-NMR (DMSO, 400 MHz):δ=9.34 (broad, 2H), 5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H),2.50-2.45 (m, 5H).

Synthesis of 516

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt₃ (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO₃solution (1×50 mL). The organic layer was then dried over anhyd. Na₂SO₄and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). 1H-NMR (CDCl3, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]-232.3 (96.94%).

Synthesis of 517A and 517B

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na₂SO₃ and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO₃ (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an Na₂SO₄ and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). ¹H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]-266.3,[M+NH4+]-283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518a

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518a (1.2 g, 41%) was obtained as a colorlessoil. 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

Synthesis of 518b

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518b (x g, y %) was obtained as a colorless oil.

Synthesis of 518c

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518c (1.2 g, 82%) was obtained as a colorlessoil. 1H-NMR (CDCl3, 400 MHz): δ=7.34-7.33 (m, 4H), 7.30-7.29 (m, 1H),5.35-5.32 (m, 6H), 5.12 (s, 2H), 4.76-4.73 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.75 (m, 5H), 2.04-1.92 (br m, 10H), 1.62 (m, 4H), 1.48 (m, 2H),1.37-1.24 (br m, 38H), 0.87-0.81 (m, 6H). HPLC-88.72%.

Synthesis of 518d

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518d (1.8 g, 77%) was obtained as a colorlessoil. 1H-NMR (CDCl3, 400 MHz): δ=7.34-7.33 (m, 4H), 7.30-7.28 (m, 1H),5.36-5.32 (m, 4H), 5.12 (s, 2H), 4.77-4.70 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.75 (m, 5H), 2.04-2.00 (m, 4H), 1.95-1.92 (m, 2H), 1.62 (m, 4H),1.56-1.53 (m, 2H), 1.35-1.23 (br m, 40H), 0.87-0.84 (m, 6H).HPLC-98.34%.

Synthesis of 518e

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518e (1.8 g, 89%) was obtained as a colorlessoil. 1H-NMR (CDCl3, 400 MHz): δ=7.34-7.33 (m, 4H), 7.31-7.28 (m, 1H),5.35-5.28 (m, 4H), 5.12 (s, 2H), 4.78-4.71 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 5H), 2.04-2.01 (m, 4H), 1.95-1.92 (m, 2H), 1.63 (m, 4H),1.48 (m, 2H), 1.35-1.24 (br m, 24H), 0.89-0.84 (m, 6H). HPLC-90.98%.

Synthesis of 518f

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518f (1.9 g, 91%) was obtained as a colorlessoil. 1H-NMR (CDCl3, 400 MHz): δ=7.34-7.33 (m, 4H), 7.31-7.27 (m, 1H),5.37-5.28 (m, 4H), 5.12 (s, 2H), 4.78-4.71 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.75 (m, 5H), 2.04-2.02 (m, 4H), 1.95-1.92 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.35-1.24 (br m, 28H), 0.89-0.84 (m, 6H). HPLC-96.44%.

General Procedure for the Synthesis of Compounds 519a-g

A solution of compound 508a-g (1 eq) in hexane (15 mL) was added in adropwise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na₂SO₄ then filtered through celite and reducedto an oil. Column chromatography provided the pure 519a-g.

Preparation of Compound 519a (ALNY-100)

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 519a (1.3 g, 68%) was obtained as a colorlessoil. ¹³C NMR δ=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7,38.3, 35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2(×3), 25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecularweight for C₄₄H₈₀NO₂ (M+H)⁺ Calc. 654.6; Found 654.6.

Preparation of Compound 519b (ALNY-189)

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518b (1.41 g, 1.83 mmol) providedcompound 519b (1.2 g, 99%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ5.48-5.25 (m, 10H), 4.60 (d, J=4.0, 2H), 2.86-2.65 (m, 8H), 2.31-2.16(m, 7H), 2.14-1.96 (m, 11H), 1.63 (dd, J=10.1, 5.9, 2H), 1.53-1.16 (m,38H), 1.02-0.91 (m, 3H), 0.91 (s, 3H).

Preparation of Compound 519c (ALNY-107)

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518c (0.92 g, 1.19 mmol) providedcompound 519c (0.7 g, 91%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ5.51-5.20 (m, 6H), 4.62 (d, J=4.7, 2H), 2.76 (dd, J=11.6, 5.4, 3H), 2.25(s, 6H), 2.17-1.85 (m, 10H), 1.63 (dd, J=10.1, 5.9, 2H), 1.56-1.15 (m,46H), 0.98-0.77 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 130.57, 130.51,130.39, 130.35, 130.33, 130.11, 130.05, 130.03, 128.14, 128.11, 112.54,79.51, 77.54, 77.22, 76.90, 64.65, 44.92, 38.53, 36.31, 35.59, 32.83,32.11, 31.73, 30.16, 30.09, 29.98, 29.92, 29.87, 29.84, 29.76, 29.74,29.68, 29.64, 29.56, 29.53, 29.51, 29.39, 29.35, 27.45, 27.41, 25.83,24.75, 23.52, 22.90, 22.79, 14.35, 14.31. Calc. mass for the C₄₄H₈₁NO₂:656.12; found 656.5.

Preparation of Compound 519d (ALNY-108)

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518d (1.58 g, 2.03 mmol) providedcompound 519d (1.14 g, 86%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 5.50-5.20 (m, 4H), 4.61 (d, J=4.7, 2H), 2.75 (dt, J=11.7, 5.9, 3H),2.24 (s, 6H), 2.08 (ddd, J=28.4, 13.7, 6.3, 6H), 1.63 (dd, J=10.1, 5.9,2H), 1.54-1.14 (m, 54H), 0.95-0.77 (m, 6H). ¹³C NMR (101 MHz, cdcl₃) δ130.39, 130.36, 130.33, 128.14, 128.12, 112.55, 79.52, 77.55, 77.23,76.91, 64.65, 44.94, 38.55, 36.32, 35.60, 32.14, 31.74, 30.17, 30.10,29.92, 29.88, 29.84, 29.79, 29.77, 29.75, 29.69, 29.59, 29.57, 29.51,27.46, 27.44, 27.41, 25.83, 24.76, 23.53, 22.91, 22.80, 14.35, 14.31.Calc. mass for the C₄₄H₈₃NO₂: 658.14; found 658.5.

Preparation of Compound 519e (ALNY-110)

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518e (1.31 g, 1.98 mmol) providedcompound 519e (0.73 g, 68%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 5.51-5.20 (m, 4H), 4.62 (d, J=4.8, 2H), 2.88-2.61 (m, 3H), 2.24 (s,6H), 2.08 (ddd, J=20.5, 13.7, 6.3, 6H), 1.64 (dd, J=10.1, 5.9, 2H),1.55-1.14 (m, 38H), 0.96-0.78 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ130.55, 130.51, 130.37, 130.33, 130.31, 130.11, 130.05, 130.02, 128.14,128.11, 112.53, 79.51, 77.54, 77.23, 76.90, 64.65, 44.92, 38.53, 36.31,35.57, 32.83, 32.11, 31.73, 30.16, 30.09, 29.98, 29.92, 29.87, 29.84,29.77, 29.74, 29.68, 29.63, 29.56, 29.53, 29.51, 29.39, 29.35, 27.44,27.40, 25.83, 24.75, 23.52, 22.90, 22.79, 14.34, 14.30. Calc. mass forthe C₃₆H₆₇NO₂: 545.9; found 646.4.

Preparation of Compound 519f (ALNY-109)

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518f (1.5 g, 2.16 mmol) providedcompound 519f (1.13 g, 92%) as a colorless oil. ¹H NMR (400 MHz, CDCl₃)δ 5.47-5.19 (m, 4H), 4.62 (d, J=4.7, 2H), 2.88-2.62 (m, 3H), 2.25 (s,6H), 2.08 (ddd, J=28.3, 13.7, 6.3, 6H), 1.64 (dd, J=10.1, 5.9, 2H),1.56-1.14 (m, 42H), 0.94-0.78 (m, 6H). ¹³C NMR (101 MHz, CDCl₃) δ130.18, 130.15, 130.12, 127.94, 127.91, 112.36, 79.31, 77.32, 77.01,76.69, 64.44, 44.68, 38.30, 36.13, 35.38, 31.92, 31.52, 29.95, 29.88,29.71, 29.65, 29.62, 29.56, 29.53, 29.47, 29.35, 29.29, 27.25, 27.22,27.20, 25.62, 24.53, 23.32, 22.69, 22.58, 14.13, 14.08. Calc. mass forthe C₃₈H₇₁NO₂: 573.9; found 574.5.

Preparation of Compound 519g

Using a procedure analogous to that described for the synthesis ofcompound 506, reduction of compound 518g (2 mmol) provided compound 519g(92%) as a colorless oil.

Example 12 Synthesis of Ketal 519

Preparation of Compound 516

An ice-cold solution of compound 513 (0.97 g, 11.5 mmol) and NEt₃ (1.2eq) in DCM (50 mL) was treated in a dropwise fashion over 5 min withMsCl (1.1 eq). After 1 h at 0° C., aqueous workup gave crude compound514 (pure by TLC) which was used without purification in the next step.

Compound 514 (11.5 mmol) was treated with 33% H₂NMe in EtOH (50 mL) over72 h. Excess H₂NMe was removed by co-evaporation with Et₂O, then theethanolic solution of compound 515 was treated with conc. HCl (2 eq).The EtOH was removed by evaporation and the residue was redissolved inDCM (100 mL). NEt₃ (6 eq) was added followed by Cbz-OSu (2.2 eq) and themixture was stirred at r.t. for 3 h. Aqueous workup then columnchromatography gave pure compound 516 as an oil (2.15 g, 81% from 513).¹H NMR (CDCl₃, 400 MHz) δ=7.40-7.28 (m, 5H), 5.70 (s, 2H), 5.14 (s, 2H),5.1-4.9 (br, m, 1H), 2.75 (s, 3H), 2.7-2.54 (br, 2H), 2.29 (d, J=18.2Hz, 2H); ¹³C NMR δ=156.2, 137.0, 129.3, 128.5, 127.9, 127.8, 67.0, 53.6,36.5, 29.7; Electrospray MS (+ve): Molecular weight for C14H18NO2 (M+H)⁺Calc. 232.1; Found 232.0.

Preparation of Compound 517

Using a procedure analogous to that described for the synthesis ofcompound 502, compound 517 (1.75 g, 71%) was obtained as a colorlessoil. ¹³C NMR δ=155.2, 137.1, 128.4, 127.8, 127.5, 127.4, 72.4, 66.1,59.8, 52.8, 34.2, 28.8, 20.8, 14.1; Electrospray MS (+ve): Molecularweight for C14H20NO4 (M+H)⁺ Calc. 266.1; Found 266.0.

Preparation of Compound 518

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (2.01 g, 63%) was obtained as a colorlessoil. ¹³C NMR δ=156.2, 136.9, 130.2 (×2), 130.1, 128.4, 128.0, 127.9(×2), 127.8, 127.7, 127.6 (×2), 112.6, 79.3, 67.0, 31.5, 29.6, 29.5(×3), 29.3 (×2), 27.2 (×3), 25.6, 24.5, 22.6, 14.1; Electrospray MS(+ve): Molecular weight for C51H84NO4 (M+H)⁺ Calc. 774.6; Found 774.6.

Preparation of Compound 519

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 519 (1.3 g, 68%) was obtained as a colorless oil.¹³C NMR δ=130.2, 130.1 (×2), 127.9 (×3), 112.3, 79.3, 64.4, 44.7, 38.3,35.4, 31.5, 29.9 (×2), 29.7, 29.6 (×2), 29.5 (×3), 29.3 (×2), 27.2 (×3),25.6, 24.5, 23.3, 226, 14.1; Electrospray MS (+ve): Molecular weight forC44H80NO2 (M+H)⁺ Calc. 654.6; Found 654.6.

Example 13 Synthesis of Ketal ALNY-102 & 103

Synthesis of 521

To a solution of 520 (2.5 g, 0.0198 moles) in toluene (10 mL) in 50 mLtwo neck RBF, were added triethylamine (3 mL, 0.02178 moles) and DPAA(Diphenylphosphoryl azide) (5.08 mL, 0.0237 moles) was added at roomtemperature. After refluxing the mixture for 2 h, Benzyl alcohol (2.5mL, 0.02376 moles) was added, and the mixture was refluxed foradditional 10 h. The resulting mixture was concentrated under vacuum,diluted with ethyl acetate and washed with 1N HCl, NaHCO3 solution andbrine respectively. The organic layer was dried over sodium sulphate andsolvent was removed under vacuum. Crude material was purified by silicagel column chromatography using 0 to 3% ethyl acetate in hexane aseluting system. (3.2 g, 57%). 1H-NMR (CDCl3, 400 MHz): δ=7.37-7.28 (m,5H), 5.67-5.64 (m, 1H), 5.59-5.55 (m, 1H), 5.08 (s, 2H), 4.77 (m, 1H),3.86 (br, 1H), 2.40-2.36 (m, 1H), 2.11 (m, 2H), 1.91-1.84 (br m, 2H),1.54 (br m, 1H).

Synthesis of 522

The compound 522 (2 g, 0.0865 moles) was dissolved in Dry DMF (20 mL) in100 mL two neck RBF and cooled to 0° C. Sodium Hydride (60% in mineraloil) (0.83 g, 0.0173 moles) was added and the mixture was stirred for 30mins under nitrogen atmosphere. Methyl iodide (4.27 g, 0.0303 moles) wasthen added slowly and the reaction mixture was then stirred at 60° C.for 4 h. The progress was monitored by TLC. After completion ofreaction, it was cooled to 0° C. and quenched with ice water (100 mL)and extracted with ethyl acetate (3×50 mL). Organic layer was separated,dried over sodium sulfate and concentrated under vacuum. Crude productwas purified by column chromatography; product was eluted with 0-10%ethyl acetate in hexane. Yield (1.8 gm, 92%). ¹H-NMR (CDCl3, 400 MHz):δ=7.35-727 (m, 5H), 5.62-5.59 (s, 2H), 5.13-5.10 (s, 2H), 4.30-4.18 (brm, 1H), 2.80 (s, 3H), 2.17-2.11 (m, 4H), 1.72-1.61 (m, 2H).

Synthesis of 523A and 523B

To a stirred solution of 522 (6 g, 0.0244 mol) in 264 mL acetone:water(10:1) in a 500 mL single neck RBF was added NMO (8.5 g, 0.0732 mol)followed by 4.6 mL solution of 6.6% OsO4 (0.311 g, 0.001224 mol) int-BuOH. Reaction was stirred for 2 hr and monitored by TLC. Aftercompletion of reaction, mixture was quenched with sat. aq. Na₂SO₃solution stirred for 30 min and extracted with DCM (300 mL×3). Organiclayer was dried over Na₂SO₄. Solvent was removed under vacuum to getcrude mix of 523A and 523B. Crude mixture was purified on 60-120 silicagel using 0-5% ethyl acetate in hexane as eluent to give purediastereomeric mixture which was separated by prep. HPLC. Peak-1 fromprep HPLC was identified as 523A. Yield-6 g (87.84%) 523B (Peak-1),Yield 3 g (68%) (Colorless solid) 1H-NMR (DMSO, 400 MHz): δ=7.39-7.30(m, 5H), 5.06 (s, 2H), 4.57-4.55 (d, 1H), 4.30-4.29 (m, 1H), 3.91-3.85(m, 1H), 3.66 (br s, 1H), 3.43-3.40 (m, 1H), 2.73 (s, 3H), 1.77-1.67 (m,3H), 1.39-1.33 (m, 2H), 1.22-1.17 (m, 1H). LC-MS:—[M+H]-280.2.HPLC:—99.85%. Stereochemistry confirmed by X-ray.

To a stirred mixture of 524A and 524B (1.2 g, 0.0043 mol) and dilinoleylketone (2.26 g, 0.0043 mol) in 150 mL toluene, p-TSA (0.073 g, 0.00043mol) was added at room temperature. The reaction mixture was heated toreflux over Soxhlet Apparatus for 4 h. After completion of reaction (byTLC), toluene was removed on rotary evaporator and residual compound wasdissolved in DCM (50 mL). Organic layer was washed with saturated NaHCO3(1×10 ml), followed by water (1×15 mL). Organic layer was separated,dried over Na2SO4 and concentrated under vacuum. Crude product waspurified on neutral Alumina using 0-5% ethyl acetate in hexane aseluent.

524A: Yield-1.2 g (36%) 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.25 (m, 5H),5.37-5.30 (m, 8H), 5.11 (s, 2H), 4.13 (m, 2H), 4.1-3.80 (m, 1H), 2.77(m, 7H), 2.22 (m, 1H), 2.05-2.00 (m, 8H), 1.9 (m, 1H), 1.71-1.63 (m,4H), 1.37-1.26 (br m, 40H), 0.89-0.85 (m, 6H). HPLC:—98.29%

524B: Yield-1.0 g (37%). 1H-NMR (CDCl3, 400 MHz): δ=7.34-7.27 (m, 5H),5.35-5.28 (m, 8H), 5.12 (br s, 2H), 4.32-4.26 (m, 2H), 4.07-4.05 (m,1H), 2.80 (s, 3H), 2.77-2.74 (m, 4H), 2.04-2.01 (m, 9H), 1.84-1.66 (m,5H), 1.35-1.15 (br m, 40H), 0.89 (m, 6H). HPLC:—95.18%.

Synthesis of ALNY-102 & ALNY-103

Prepared the compound ALNY-102 and 103 by following similar procedure ascompound 506.

The N-Cbz-compound 524B (0.9 g, 1.14 mmol) and 1M solution of LAH in THF(2.28 mL, 2.28 mmol, 2.0 eq), gave 0.65 g (85%) of the pure productALNY-103. ¹H NMR (400 MHz, CDCl₃) δ 5.48-5.22 (m, 8H), 4.33 (dd, J=9.5,4.1, 1H), 4.23-4.00 (m, 1H), 2.77 (t, J=6.4, 4H), 2.65 (s, 1H), 2.34 (s,6H), 2.18-1.95 (m, 9H), 1.86 (dd, J=11.7, 5.7, 3H), 1.78-1.59 (m, 4H),1.54 (d, J=8.3, 2H), 1.45-1.16 (m, 37H), 0.98-0.79 (m, 6H). ¹³C NMR (101MHz, CDCl₃) δ 130.40, 130.35, 128.16, 128.13, 111.45, 77.54, 77.23,76.91, 73.11, 72.94, 57.93, 54.76, 41.69, 38.22, 36.23, 32.09, 31.74,30.21, 29.91, 29.74, 29.71, 29.57, 29.54, 29.02, 27.45, 27.41, 26.89,25.84, 24.71, 24.38, 23.34, 22.79, 21.42, 14.30. Calc. mass for theC₄₅H₈₁NO₂: 668.13; found 668.5.

Example 14 Synthesis of ALNY-169

Synthesis of 525

The compound 522 (7 g, 0.02857 moles) was dissolved in dry DCM (60 mL)in a 250 ml two neck RBF and cooled to 0° C. After stirring the mixturefor 10 minutes (50-75%) m-CPBA (15.66 g, 0.04571 moles) was added inportions over a span of 30 minutes. Reaction mixture was then warmed toambient temperature and stirred for 4 h. Progress of the reaction wasmonitored by TLC. After completion of the reaction, mixture was dilutedwith ethyl acetate (250 ml) and washed with sat. Na₂SO₃ solution (3×50mL) followed by sat. NaHCO₃ solution (2×100 ml). Organic layer was thenwashed with brine (1×50 mL), dried over an. Na₂SO₄, and concentrated invacuum. Crude mass thus obtained was purified by silica gel (60-120mesh) column chromatography using 3% methanol in dichloromethane to givepure compound 525 as a thick liquid. Yield: 4.32 gm (58%). ¹H NMR (400MHz, CDCl₃) δ=7.36-7.25 (s, 5H), 5.11 (s, 2H), 4.12-3.88 (m, 1H), 3.12(br, 2H), 2.75 (s, 3H), 2.25-2.21 (m, 1H), 2.05 (m, 1H), 1.92-1.82 (m,2H), 1.61-1.57 (m, 1H), 1.33-1.30 (m, 1H). LCMS [M⁺H]: 279.3.

Synthesis of 526

A single neck 250 ml RBF charged with Compound 525 (4.32 g, 0.0165moles) was cooled to 0° C., and then dissolved in 50 mL 2% aq. H₂SO₄.After stirring for 10 minutes, reaction mixture was warmed to roomtemperature and stirred for 1 h. Reaction was monitored by TLC. Aftercompletion of the reaction, mixture was cooled again and basified with6N NaOH. Aqueous solution was then extracted with ethyl acetate (50ml×3) and organic layer was separated, dried and evaporated on rotaryevaporator. Crude compound thus obtained was purified by 230-400 meshflash silica gel column chromatography using 0-2% methanol indichloromethane as eluting system to afford the sticky mass as adiastereomeric mixture. Yield: ¹H NMR (400 MHz, DMSO) δ=7.38-7.28 (m,5H), 5.06 (s, 2H), 4.75 (s, 1H), 4.62-4.61 (d, 1H), 4.24 (br, 1H), 3.71(s, 1H), 3.48 (s, 1H), 2.71 (s, 3H), 1.84 (m, 1H), 1.76-1.74 (m, 2H),1.52-1.50 (m, 1H), 1.37 (s, 1H), 1.21-1.18 (m, 1H). LCMS [M+H]: 280.3,[M+NH₄]: 297.3; HPLC shows a mixture of two diasteromers in 50:50 ratio.

Synthesis of 527

A 100 ml single neck RBF was charged with diastereomeric mixture of 526(0.6 g, 0.00215 mol), dimethyl acetal of dilinoleyl ketone (1.23 g,0.002150 mol) 50 ml toluene, catalytic amount of p-TSA. The resultingmixture was then stirred and heated to reflux vigorously over a Soxhletapparatus containing 4A molecular sieves and 50 ml toluene. Progress ofthe reaction was monitored by TLC. After 6 h mixture was cooled to roomtemperature, volatilities were removed under vacuum and residual massstirred in n-hexane for 10 minutes then decanted off. This procedure wasrepeated twice to remove the unreacted diol. All the hexane washingswere combined and concentrated to give crude material which was purifiedby column chromatography on neutral alumina using 3% ethyl acetate inn-hexane as eluent to afford Alny-8-015 which was confirmed by ¹H NMRand MS. Yield: 0.6 g. (37.5%) ¹H NMR (400 MHz, CDCl₃) δ=7.35-7.30 (m,5H), 5.40-5.29 (m, 8H), 5.15-5.07 (dd, 2H), 4.48 (m, 1H), 3.56-3.53 (m,1H), 3.41-3.36 (m, 1H), 2.96 (s, 3H), 2.77-2.74 (m, 4H), 2.30-2.27 (m,1H), 2.14 (m, 1H), 2.06-2.01 (m, 8H), 1.70 (m, 3H), 1.61 (m, 4H),1.35-1.24 (m, aliphatic protons), 0.89-0.859 (m, 6H). Mass-[M+H]-788.8 &[M+Na]-811.8 HPLC purity-97.32%.

Synthesis of ALNY-169

Prepared the ALNY-169 by following similar procedure as compound 506,using N-Cbz-compound of 527 (0.52 g, 0.66 mmol) and 1M solution of LAHin THF (1.32 mL, 1.32 mmol), gave 0.4 g (91%) of the pure productALNY-169. ¹H NMR (400 MHz, CDCl₃) δ 5.50-5.22 (m, 8H), 3.67 (ddd,J=12.1, 8.9, 3.3, 1H), 3.39-3.16 (m, 1H), 2.77 (t, J=6.4, 4H), 2.44 (dd,J=12.7, 2.2, 1H), 2.29 (s, 1H), 2.24 (s, 6H), 2.06 (dt, J=13.5, 10.9,8H), 1.99-1.87 (m, 1H), 1.63 (dd, J=16.1, 9.8, 5H), 1.51-1.14 (m, 39H),0.89 (t, J=6.7, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 130.40, 128.14, 112.25,81.03, 77.54, 77.22, 76.91, 75.60, 61.55, 44.11, 38.19, 38.14, 31.74,30.24, 30.21, 29.89, 29.80, 29.77, 29.74, 29.57, 29.54, 27.46, 27.41,25.84, 24.86, 24.36, 24.23, 22.80, 14.31. Calc. mass for the C₄₅H₈₁NO₂:668.1; found 668.5.

Example 15 Synthesis of Ketal ALNY-115 & 116

Preparation of Compound 651

An ice-cold solution of compound 650 (1.05 g, 10.7 mmol) and NEt₃ (1.2eq) in DCM (50 mL) was treated in a dropwise fashion over 5 min withMsCl (1.1 eq). After 1 h at 0° C., aqueous workup gave crude mesylate(1.88 g, 99%, pure by TLC) which was used without purification in thenext step. The mesylate (10.5 mmol) was treated with 33% H₂NMe in EtOH(100 mL) at 50° C. over 18 h. Excess H₂NMe was removed by co-evaporationwith Et₂O, then the ethanolic solution of the product was treated withconc. HCl (2 eq). The EtOH was removed by evaporation and the residuewas redissolved in DCM (100 mL). NEt₃ (4 eq) was added followed byCbz-OSu (1.5 eq) and the mixture was stirred at r.t. for 3 h. Aqueousworkup then column chromatography gave pure compound 651 as an oil (86%from 650). ¹H NMR (400 MHz, CDCl₃) δ 7.54-7.04 (m, 5H), 5.65 (d, J=8.6,2H), 5.13 (s, 2H), 3.27 (t, J=8.9, 2H), 2.94 (s, 3H), 2.60 (s, 1H),2.51-2.28 (m, 2H), 2.04 (dd, J=22.1, 15.3, 2H); ¹³C NMR (101 MHz, CDCl₃)δ 129.47, 128.40, 127.83, 127.75, 66.92, 53.74, 53.13, 38.92, 36.34,36.23, 36.06, 35.78; Electrospray MS (+ve): Molecular weight forC15H19NO2 (M+H)⁺ Calc. 246.1; Found 246.0.

The diols 652A and 652B were synthesized using our standard reactionconditions starting from the commercially available cyclopentene alcohol650.

Synthesis of Compounds 652A and 652B

Compound 652A and compound 652B were successfully separated from thediastereomeric mixture by preparative HPLC method. (the two isomers werein the ratio of 88:12) LC-MS-[M+H]-280.4 & [M+NH₄ ⁺]-297.5.

652A: ¹H-NMR (400 MHz, DMSO), δ=7.38-7.30 (m, 5H), 5.05 (s, 2H), 4.34(d, 2H), 3.83 (br, 2H), 3.12-3.10 (m, 2H), 2.84-2.80 (m, 3H), 1.60-1.56(m, 2H), 1.34-1.33 (m, 2H) HPLC purity-99.78%.

652B: ¹H-NMR (400 MHz, DMSO), δ=7.36-7.31 (m, 5H), 5.05 (s, 2H), 4.33(br, 2H), 3.73 (m, 2H), 3.25-3.20 (m, 2H), 2.85-2.82 (m, 3H), 2.07-2.03(m, 1H), 1.82-1.73 (m, 2H), 1.28-1.25 (m, 2H) HPLC purity-98.76%.

Using a procedure analogous to that described for the synthesis ofcompound 505, compounds 653A and 653B were obtained as yellow oil.

653A:

Yield: 2.1 g (67.74%) colorless oil ¹H-NMR (400 MHz, CDCl₃), δ=7.33-7.29(m, 5H), 5.38-5.28 (m, 8H), 5.10 (s, 2H), 4.58-4.55 (m, 2H), 3.29-3.23(m, 2H), 2.92 (m, 3H), 2.77-2.74 (m, 4H), 2.5 (m, 1H), 2.06-2.01 (m,8H), 1.92-1.88 (m, 2H), 1.60 (m, 2H), 1.45 (m, 2H), 1.37-1.25 (m,aliphatic protons), 0.89-0.85 (m, 6H) HPLC purity:—98.95%

653B:

Yield 0.210 g, (62.00%) ¹H-NMR (400 MHz, CDCl₃), δ=7.33 (m, 5H),5.37-5.32 (m, 8H), 5.10 (s, 2H), 4.57 (br, 2H), 3.35-3.33 (m, 2H), 2.95(s, 3H), 2.77-2.74 (m, 4H), 2.33 (m, 1H), 2.26 (m, 1H), 2.06-2.00 (m,8H), 1.92 (m, 2H), 1.65 (m, 2H), 1.60 (m, 2H), 1.37-1.25 (m, aliphaticprotons), 0.89-0.85 (m, 6H) HPLC purity:—98.84%

Preparation of ALNY-115

Prepared the compound ALNY-115 by following similar procedure ascompound ALNY-100, using N-Cbz-compound 653A (0.21 g, 0.27 mmol, 1.0 eq)and 1M solution of LAH in THF (0.4 mL, 0.4 mmol, 1.5 eq), gave 0.09 g(50%) of the pure product ALNY-115. ¹H NMR (400 MHz, CDCl₃) δ 5.53-5.16(m, 8H), 4.68-4.46 (m, 2H), 2.79 (t, J=6.6, 4H), 2.37 (d, J=7.1, 2H),2.24 (d, J=5.1, 6H), 2.16-1.94 (m, 11H), 1.73-1.64 (m, 2H), 1.58 (ddd,J=20.2, 10.4, 6.8, 4H), 1.48-1.21 (m, 37H), 0.91 (dd, J=8.8, 5.1, 6H).¹³C NMR (101 MHz, CDCl₃) δ 130.19, 130.17, 130.15, 127.95, 127.94,127.93, 115.27, 80.90, 77.34, 77.02, 76.70, 65.01, 45.83, 37.40, 37.10,31.55, 31.54, 31.53, 29.99, 29.70, 29.68, 29.58, 29.56, 29.50, 29.36,29.35, 29.32, 27.26, 27.24, 27.21, 25.64, 24.42, 23.83, 22.59, 14.10.Calc. mass for the C₄₅H₈₁NO₂: 668.1; found 668.5.

Preparation of ALNY-116

Prepared the compound ALNY-116 by following similar procedure ascompound ALNY-100, using N-Cbz-compound of the corresponding product(2.1 g, 2.66 mmol, 1.0 eq) and 1M solution of LAH in THF (4.0 mL, 4.0mmol, 1.5 eq), gave 1.2 g (68%) of the pure product ALNY-116.

¹H NMR (400 MHz, CDCl₃) δ 5.57-5.21 (m, 8H), 4.67-4.53 (m, 2H), 2.79 (t,J=6.5, 4H), 2.52-2.33 (m, 1H), 2.23 (m, 8H), 2.14-1.94 (m, 10H), 1.66(dd, J=10.1, 6.1, 2H), 1.58-1.22 (m, 39H), 1.22-1.07 (m, 2H), 0.91 (t,J=6.9, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 130.19, 130.17, 130.14, 127.96,127.94, 127.93, 112.24, 80.36, 77.34, 77.02, 76.70, 64.27, 45.80, 38.75,36.15, 35.85, 34.67, 31.54, 30.01, 29.88, 29.71, 29.69, 29.68, 29.58,29.53, 29.49, 29.37, 29.36, 29.31, 27.27, 27.24, 27.21, 25.64, 24.63,23.17, 22.59, 22.58, 14.10. Calc. mass for the C₄₅H₈₁NO₂: 668.1; found668.5.

Example 16 Synthesis of ALNY-121 and ALNY-122

Synthesis of 654A and 654B

To a solution of compound 640 (5 g, 0.01930 mol) in a mixture of (10:1)mixture of acetone and water (220 ml) in a single neck 500 ml RBF wasadded NMO (6.784 g, 0.05791 mol) followed by a 6.25% solution of OsO₄(0.245 g, 0.0009652 mol) in tert-butanol. The resulting solution wasstirred at room temperature and progress of the reaction was monitoredby TLC. After complete consumption of compound 640 (˜2.0 h by TLC)reaction mixture was quenched with 100 ml aqueous Na₂SO₃ solution andthe mixture was stirred at room temperature for 1.0 h. Aqueous phase wasthen extracted well with dichloromethane (2×250 ml) and separated out.Organic layer was washed with sat. NaHCO₃ (2×50 ml) followed by brine(1×50 ml). Organic phase was then dried over Na₂SO₄, filtered andconcentrated in vacuum to give crude mass, which was subjected to columnpurification on 60-120 mesh silica gel and eluted with 6-10% methanol indichloromethane system to afford pure diastereomeric mixture as thickyellow oil. Yield: 5.7 g, (100%) ¹H-NMR (400 MHz, CDCl³), δ=7.34-7.27(m, 5H), 5.12 (s, 2H), 3.98-3.92 (m, 1H), 3.61-3.57 (m, 1H), 3.17-3.08(m, 2H), 2.92-2.90 (m, 3H), 2.16-1.84 (m, 4H), 1.68 (m, 2H), 1.40-1.37(m, 1H) LC-MS: [M+H]-294.3, HPLC purity: two peaks in a ratio (15:85).

Diastereomers were separated by preparative HPLC.

Peak-1:

¹H-NMR (400 MHz, CDCl₃), δ=7.35-7.28 (m, 5H), 5.10 (s, 2H), 3.98-3.94(m, 1H), 3.63-3.55 (m, 1H), 3.19-3.06 (m, 2H), 2.90 (s, 3H), 2.00-1.99(m, 1H), 1.90-1-59 (m, 4H), 1.23-1.17 (m, 1H), 1.12-0.86 (m, 2H), LC-MS:[M+H]-294.3 & [M+NH₄ ⁺]-311.4, HPLC purity: 100%

Peak-2:

¹H-NMR (400 MHz, CDCl₃), δ=7.32-7.29 (m, 5H), 5.12 (s, 2H), 3.93 (br,1H), 3.65-3.14 (m, 2H), 2.90 (s, 3H), 1.91 (m, 1H), 1.67-1.48 (m, 2H),1.41-1.26 (m, 5H), LC-MS: [M+H]-294.4& [M+Na]-316.4, HPLC purity: 99.36%

Synthesis of 655A and 655B General Procedure:

A mixture of diol (1 eq), dilinoleyl ketone (3 eq), 50 ml toluene andcatalytic amount of p-TSA (0.1 eq) in a 100 ml single neck RBF werestirred and heated to reflux vigorously over Soxhlet apparatuscontaining 4A molecular sieves and 50 ml toluene. Progress of thereaction was monitored by TLC. After completion of the reaction (˜12-14h), mixture was cooled to room temperature and then toluene was removedin vacuum to give crude mass which was purified by column chromatographyon neutral alumina using 3-5% ethyl acetate in hexane as eluting systemto afford pure product as yellow oil.

Peak-1 655A

Yield 0.3 g (38%) ¹H-NMR (400 MHz, CDCl₃), δ=7.35-7.28 (m, 5H),5.40-5.28 (m, 8H), 5.10 (s, 2H), 4.23-4.19 (m, 1H), 4.07-4.02 (m, 1H),3.19-3.06 (m, 2H), 2.91-2.89 (m, 3H), 2.77-2.75 (m, 4H), 2.05-2.00 (m,8H), 1.98 (m, 1H), 1.85 (m, 1H), 1.72-1.65 (m, 3H), 1.37-1.24 (m,aliphatic protons), 0.89-0.85 (m, 6H). HPLC purity: 90.88%

Peak-2 (655B)

Yield 0.225 g (38%) ¹H-NMR (400 MHz, CDCl₃), δ=7.34-7.29 (m, 5H),5.39-5.28 (m, 8H), 5.10 (s, 2H), 4.14 (br, 1H), 4.02 (m, 1H), 3.22-3.17(m, 1H), 3.09-3.05 (m, 1H), 2.91 (1, 3H), 2.77-2.74 (m, 4H), 2.05-2.00(m, 8H), 1.80 (m, 1H), 1.65 (m, 3H), 1.35-1.26 (m, aliphatic protons),0.89-0.85 (m, 6H). HPLC purity: 92.83%

Preparation of Compound ALNY-121

Prepared the compound ALNY-121 by following similar procedure ascompound ALNY-100, using N-Cbz-compound of the corresponding product(0.28 g, 0.35 mmol, 1.0 eq) and 1M solution of LAH in THF (0.52 mL, 0.52mmol, 1.5 eq), gave 0.15 g (66%) of the pure product ALNY-121. ¹H NMR(400 MHz, CDCl₃) δ 5.48-5.18 (m, 8H), 4.29-4.16 (m, 1H), 4.06 (dt,J=8.3, 5.6, 1H), 2.76 (t, J=6.4, 4H), 2.18 (s, 6H), 2.14-1.91 (m, 11H),1.90-1.60 (m, 5H), 1.60-1.46 (m, 3H), 1.46-1.13 (m, 38H), 0.94-0.76 (m,6H). ¹³C NMR (101 MHz, cdcl₃) δ 130.40, 130.38, 128.16, 111.29, 77.54,77.23, 76.91, 74.02, 73.13, 66.44, 46.15, 38.44, 36.58, 32.07, 31.75,30.25, 29.92, 29.90, 29.78, 29.75, 29.72, 29.57, 29.55, 29.54, 28.82,28.52, 27.47, 27.43, 26.03, 25.86, 24.74, 24.42, 22.79, 14.29 Calc. massfor the C₄₆H₈₃NO₂: 682.1; found 682.5.

Preparation of ALNY-122

Using a similar procedure as described for the synthesis of 653B, thesix membered analog 655B was synthesized and purified.

ALNY-122 was prepared by following similar procedure as compoundALNY-100, using N-Cbz-compound of the corresponding product (0.21 g,0.26 mmol, 1.0 eq) and 1M solution of LAH in THF (0.4 mL, 0.4 mmol, 1.5eq), gave 0.08 g (50%) of the pure product 11. ¹H NMR (400 MHz, CDCl₃) δ5.50-5.19 (m, 8H), 4.16 (dd, J=8.2, 4.2, 1H), 4.05 (dt, J=9.7, 6.1, 1H),2.76 (t, J=6.4, 4H), 2.16 (d, J=10.3, 6H), 2.10-1.97 (m, 10H), 1.95-1.83(m, 1H), 1.76-1.48 (m, 7H), 1.48-1.19 (m, 39H), 0.88 (t, J=6.8, 6H). ¹³CNMR (101 MHz, cdcl₃) δ 130.40, 130.38, 128.17, 111.50, 77.55, 77.23,76.91, 74.33, 73.21, 66.44, 46.17, 38.75, 36.87, 34.64, 32.36, 31.75,30.25, 29.92, 29.90, 29.77, 29.75, 29.72, 29.58, 29.54, 27.48, 27.47,27.43, 26.70, 25.86, 25.12, 24.72, 24.54, 22.80, 14.29. Calc. mass forthe C₄₆H₈₃NO₂: 682.1; found 682.5.

Example 17 Synthesis of ALNY-144

Synthesis of Compound 671

D (−) ribose (15 g, 0.1 mmol) was dissolved in 300 ml methanol in a 1liter single neck RBF and cooled to 0° C. under nitrogen atmosphere. Tothis stirred solution was added conc. H2SO4 (2 mL) slowly and thenreaction mixture was warmed to room temperature and stirred for 20 h.Progress of the reaction was monitored by TLC. After completion ofreaction (˜20 h), reaction mixture was concentrated under vacuum to halfthe original volume. Sodium carbonate (15 g) was then added into it andmixture was stirred for 2 h. Mixture was then filtered off and filtratewas concentrated to get compound 671 as pale yellow oil. Yield: 20 gcrude (matched with authentic procured from Aldrich).

Synthesis of Compound 672

Compound 671 (2 g, 0.0121 mmol) was dissolved in 40 ml anhydrous DMF ina 100 mL two neck RBF and cooled to 0° C. To this stirred solution (60%as oil suspension) NaH (2.18 g, 0.0455 mmol) was added in portions undernitrogen atmosphere and mixture was stirred for 35 min. Benzyl bromide(5.32 mL, 0.0445 mmol) was then added slowly over a period of 30 min.Reaction mixture was then warmed to room temperature and stirring wascontinued for 16 h at room temperature. The progress was monitored byTLC. Upon completion, reaction mixture was poured onto crushed ice (˜200g). Aqueous phase was well extracted with Ethyl acetate (2×100 ml).Organic layer was then dried over Na₂SO₄, and ethyl acetate was strippedoff to afford crude compound which was purified by column chromatographyusing 60-120 mesh silica gel with 0-30% EtOAc:Hexane as eluting systemto get compound 2 as pale yellow liquid. Yield: 3 g (57%). 1H-NMR(CDCl3, 400 MHz): δ=7.36-7.24 (m, 15H), 4.9 (s, 1H), 4.67-4.51 (m, 5H),4.45-4.42 (m, 1H), 4.34-4.32 (m, 1H), 4.01-3.98 (m, 1H), 3.81 (m, 1H),3.61-3.59 (m, 1H), 3.58-3.47 (m, 1H), 3.3 (s, 3H). LC-MS: [M+H]-435.3present.

Synthesis of Compound 673

A single neck 100 ml RBF was charged with Compound 672 (3 g, 0.0069mmol) and 80% aq.AcOH solution. Resulting mixture was then heated at 90°C. for 9 h and monitored by TLC. Reaction mixture was cooled to roomtemperature and concentrated on rotatory evaporator. Residual mass wasagain co-evaporated with toluene and methanol to remove traces of AcOH.To this residue methanol (35.3 ml) was added and stirred for some timeat 0° C. This was followed by addition of NaBH4 (0.277 g, 0.00734 mmol)in portions and mixture was stirred at 0° C. for 1 h. Solvent wasevaporated off, residue was diluted with water (20 ml) and compound wasextracted with DCM (2×20 ml). Organic layer was washed with 10% sodiumcarbonate, dried over Na2SO4 and concentrated in vacuum. Crude compoundthus obtained was purified by silica gel (60-120 mesh) columnchromatography using (0-50%) ethyl acetate & n-hexane as eluent to getcompound 673 as a thick liquid. Yield: 1.7 g (59%). 1H-NMR (CDCl3, 400MHz): δ=7.35-7.23 (m, 15H), 4.73 (m, 1H), 4.65-4.56 (m, 3H), 4.50 (m,2H), 4.00-3.99 (m, 1H), 3.84-3.75 (m, 4H), 3.59-3.57 (m, 2H). LC-MS:[M+H]-423.2 present.

Synthesis of Compound 674

To a solution of Compound 673 (1.7 g, 0.00403 mmol) in 20 ml dioxane in100 ml RBF, PPh3 (2.1 g, 0.00806 mmol) was added and reaction mixturewas stirred for 5-10 minutes under nitrogen atmosphere. DEAD (1.24 ml,)was then added to this solution over a period of 1 h and reaction wascontinued at room temperature for 20 h. Progress of the reaction wasmonitored by TLC. After completion of the reaction, solvent was removedunder vacuum and residue obtained was purified by (60-120 mesh) silicagel column chromatography using (0-30%) ethyl acetate in hexane aseluting system to get compound 674 in pure forms. Yield: 1.3 g (80%).1H-NMR (CDCl3, 400 MHz): δ=7.34-7.19 (m, 15H), 4.63-4.53 (m, 4H),4.50-4.46 (m, 2H), 4.16 (m, 1H), 4.02-3.90 (m, 4H), 3.63-3.59 (dd, 1H),3.50-3.48 (dd, 1H). LC-MS: [M+H]-405.2, [M+NH4]-422.3

Synthesis of Compound 675

To a stirred solution of Compound 674 (1.3 g, 0.00321 mmol) in methanol(22 ml) and cyclohexene (7.5 ml) in a single neck RBF was addedcarefully Pd(OH)2/C (0.361 g) at once under nitrogen atmosphere and thenreaction mixture was heated to reflux at 80° C. for 18 h. Aftercompletion of the reaction (by TLC) mixture was cooled to roomtemperature and the catalyst was removed by filtration, filtrate wasconcentrated to give crude compound which was purified by columnchromatography using 60-120 mesh silica gel with (0-20%) ethyl acetatein hexane as eluting system to yield compound 675. Yield: 0.280 g (65%).¹H-NMR (DMSO, 400 MHz): δ=4.72-4.71 (m, 2H), 4.62-4.60 (m, 1H),3.98-3.93 (m, 1H), 3.84-3.81 (m, 1H), 3.76-3.72 (q, 1H), 3.58-3.55 (m1H), 3.50-3.46 (m, 2H), 3.37 (m, 1H).

Synthesis of Compound 676

Compound 675 (0.25 g, 0.00166 mmol) was dissolved in 2,2-dimethoxypropane (0.516 ml, 0.00365 mol) and acetone (2 ml) in a 25 ml singleneck RBF and cooled to 0° C. To this stirred solution 70% aq.HClO4(0.016 ml, 0.00166 mmol) solution was added slowly and after completeaddition reaction mixture was warmed to ambient temperature and stirredfor 4 h. After completion of the reaction by TLC, reaction mixture wasneutralized by solid sodium carbonate and volatilities were removed onrota vapour. DCM (20 ml) was added to the residue and mixture wasstirred for some time and DCM layer was washed with water (10 ml)followed by brine (5 ml). Organic layer was then dried over anhydrousNa2SO4 and concentrated in vacuum to give compound 676 as thick liquid.Yield: 0.270 g (94%). 1H-NMR (CDCl3, 400 MHz): δ=4.82-4.78 (m, 1H),4.60-4.58 (m, 1H), 4.13-4.10 (m, 1H), 3.99. 3.92 (m, 2H), 3.66-3.58 (m,2H), 1.76-1.73 (m 1H), 1.51-(s, 3H), 1.33 (s, 3H).

Synthesis of Compound 677

Compound 676 (0.250 g, 0.001436 mmol) was dissolved in anhydrous DCM ina 25 ml single neck RBF and cooled to 0° C. To this mixture TEA (0.5 ml,0.00359 mmol) was added under nitrogen atmosphere and mixture wasstirred for 30 min. Ms-Cl (0.28 ml, 0.00359 mmol) was then added slowlyat 0° C. and mixture was stirred at room temperature for 2 h. Aftercompletion of reaction (by TLC), reaction mixture quenched with water(20 ml) stirred for some time and DCM layer was separated out. Aqueousphase was further extracted with DCM (2×10 ml). Combined organicextracts was dried and concentrated in vacuum to give crude compoundwhich was used as such for next step. Yield: 0.360 g crude. 1H-NMR(CDCl3, 400 MHz): δ=4.83-4.82 (m, 1H), 4.67-4.65 (m, 1H), 4.30-4.20 (m,3H), 4.00-3.95 (m, 2H), 3.05 (s, 3H), 1.50 (s, 3H), 1.33 (s, 3H). LC-MS:[M+H]-253.0 & [M+NH4+]-270.0 present. The crude compound (0.360 g) and15 ml (25%) solution of methyl amine in ethanol were heated together ina 50 ml sealed tube at 50° C. for 18 h. Progress of the reaction wasmonitored by TLC. After completion of reaction, the mixture wasconcentrated under vacuum and co evaporated with diethyl ether to getcompound 677 as gummy mass. Yield: 0.5 g crude. 1H-NMR (CDCl3, 400 MHz):δ=4.82-4.80 (m, 1H), 4.51-4.49 (m, 1H), 4.43-4.39 (m, 1H), 4.01 (d, 2H),2.94 (s, 2H), 2.71 (s, 3H), 1.48 (s, 3H), 1.30 (s, 3H). LC-MS:[M+H]-188.2 present.

Synthesis of Compound 678

Crude compound 677 (0.5 g, 0.00267 mmol)) was dissolved in a mixture of(6 ml) dioxane: H2O (1:1) in a 50 ml single neck RBF and cooled to 0° C.Sodium bicarbonate (0.560 g, 0.00667 mmol) was then added in portionsand resulting mixture was stirred for 30 min. To this cold solution, asolution of Cbz-Cl (50%) in toluene (1.2 ml, 0.0040 mmol) was addedslowly. RM was then allowed to come to room temperature and stirred for2-3 h. The progress was monitored by TLC. After completion of reaction,mixture was diluted with water (10 ml) and DCM (20 ml), stirred for 10minutes and DCM layer was separated out. Aqueous phase was backextracted with DCM (1×10 ml). Organic phase was then dried over an.Na2SO4 and concentrated in vacuum to afford crude compound 678 which waspurified by column chromatography using neutral alumina using 10% ethylacetate in hexane as eluent to give pure compound 678. Yield: 0.3 g(35%). 1H-NMR (CDCl3, 400 MHz): δ=7.35-7.28 (m, 5H), 5.11 (m, 2H),4.82-4.23 (m, 2H), 4.18 (m, 1H), 3.98-3.62 (m, 2H), 3.33-3.25 (m, 2H),2.97 (s, 3H), 1.49 (m, 3H), 1.31 (m, 3H) (note: NMR reflects thepresence of rotamers) LC-MS: [M+H]-322.4 present.

Synthesis of Compound 679

A single neck 25 ml RBF was charged with compound 678 (0.3 g, 0.000934mmol) and 7 ml 70% aqueous acetic acid and the resulting mixture washeated to reflux for 3 h. After completion of reaction, solvent wasstripped off and traces of AcOH were removed by co evaporation withtoluene and methanol to get crude compound 679. Yield: 0.220 g (83%).1H-NMR (DMSO, 400 MHz): δ=7.36-7.30 (m, 5H), 5.09 (m, 2H), 4.84-4.81 (m,2H), 3.98-3.95 (m, 1H), 3.89-3.86 (m, 1H), 3.72 (m, 2H), 3.62 (m, 2H),3.52-3.48 (m, 3H). LCMS: [M+H]-282.0 & [M+NH4+]-299.3 present.

Synthesis of 680

To a mixture of Compound 679 (0.21 g, 0.000747 mmol) in toluene (10 ml)was added dilinoleyl ketone (0.433 g, 0.000822 mmol) followed bycatalytic amount of pTSA (0.014 g 0.1 eq,). Resulting mixture was thenheated to reflux for 2-3 h over soxhlet apparatus containing 4Amolecular sieves and 50 ml toluene. Progress of the reaction wasmonitored by TLC. After completion of reaction, toluene was removed onrotavapor and crude mass was dissolved in DCM (50 ml) and washed withaq. saturated bicarbonate solution (1×10 ml) followed by water (1×10ml). Organic layer was dried over an. Na2SO4 and concentrated in vacuum.Crude compound thus obtained was purified by column chromatography usingneutral alumina and eluted with 2% ethyl acetate in hexane system toyield the title compound 680. Yield: 0.34 g (60%). 1H-NMR (CDCl3, 400MHz): δ=7.35-7.29 (m, 5H), 5.40-5.28 (m, 8H), 5.11 (s, 2H), 4.78-4.40(m, 2H), 4.24-4.19 (m, 1H), 3.99-3.69 (m, 2H), 3.30-3.24 (m, 2H), 2.97(s, 3H), 2.76 (m, 4H), 2.06-2.01 (m, 8H), 1.65 (m, 2H), 1.35-1.26 (m,aliphatic protons from linoleyl chain), 0.87 (t, 6H). LC-MS: [M+H]=790.4& [M+Na]-812.4 present HPLC purity—98.87%.

Synthesis of ALNY-144

Prepared the compound ALNY-144 by following similar procedure ascompound ALNY-100, using N-Cbz-compound 680 of the corresponding product(0.3 g, 0.38 mmol, 1.0 eq) and 1M solution of LAH in THF (0.76 mL, 0.76mmol, 1.5 eq), gave 0.17 g (69%) of the pure product ALNY-144. ¹H NMR(400 MHz, CDCl₃) δ 5.54-5.22 (m, 8H), 4.76 (dd, J=7.5, 3.0, 1H), 4.50(dd, J=6.4, 1.8, 1H), 4.18-4.08 (m, 1H), 3.95 (dd, J=10.6, 1.4, 1H),3.84 (dd, J=10.6, 4.3, 1H), 2.77 (t, J=6.4, 4H), 2.27 (s, 6H), 2.04 (q,J=6.8, 8H), 1.68 (dd, J=9.9, 6.2, 2H), 1.53 (d, J=8.7, 2H), 1.49-1.17(m, 38H), 0.89 (t, J=6.8, 6H). Calc. mass for the C₄₄H₇₉NO₃: 670.1;found 670.5.

Example 18 Synthesis of ALNY-151

Synthesis of Compound 601

To suspension of compound 600 (10 gm, 0.0866 moles) in 2,2-dimethoxypropane (20.73 ml, 0.1399 moles) and dry acetone (140 ml) in a 500 mltwo neck RBF was added 60% perchloric acid (4.5 ml, 0.066 moles) dropwise over 5 mins at 5° C. The reaction mixture was warmed to roomtemperature and stirred for 2 h. Methanol (14 ml) was then added andsolution was stirred for an additional 2 h. Progress was monitored byTLC. The solution was then cooled at 5° C. and neutralized with aq.sodium carbonate solution (4 gm in 15 ml of water). Mixture was filteredoff and filtrate was concentrated on rotary evaporator to give yellowsolid that was dissolved in diethyl ether (200 ml), washed with water(150 ml) followed by brine (150 ml), dried over Na₂SO₄, and concentratedin vacuum to afford colorless oil. Crude oil was distilled under highvacuum (85-89° C., 0.25 mm of mercury) to give 601 as a colorlessliquid.

Yield: (40%). ¹H NMR (400 MHz, CDCl3) δ=4.95 (s, 1H), 4.81-83 (d, 1H),4.56-4.58 (d, 1H), 4.42 (bs, 1H), 3.33-3.7 (m, 1H), 3.57-3.63 (m, 1H),3.42 (s, 3H), 3.22-3.26 (m, 1H), 1.47 (s, 3H), 1.3 (s, 3H).

Synthesis of Compound 602

To a stirred solution of 601 (1 gm, 0.00490 moles) in dichloromethane(12 ml) and TEA (1.23 ml, 0.01225 moles) in a 50 ml two neck RBF at 0°C., was added methanesulfonyl chloride (0.95 ml, 0.01225 moles) dropwise over 5 min under nitrogen atmosphere. Reaction mixture was thenwarmed to room temperature and continued for 5 h. Progress was monitoredby TLC. After completion of the reaction, mixture was washed withsaturated sodium bicarbonate (2×7 ml) followed by water (2×7 ml).Organic layer was dried over Na₂SO₄ and solvent was evaporated off togive crude compound which was used for the next step without anypurification. Yield: (1.05 gm, 85%). ¹H NMR (400 MHz, CDCl₃) δ 4.97(s1H), 4.67-4.69 (d, 1H), 4.58-4.59 (d, 1H), 4.37-4.41 (t, 1H), 4.19 (d,2H), 3.32 (s, 3H), 3.11 (s, 1H), 3.05 (s, 3H), 1.46 (s, 3H), 1.30 (s,3H). LCMS [M⁺H]: 283.2. A solution of the crude product (1.05 g,0.003723 moles) in 10 ml (25%) methylamine in ethanol was stirred at 50°C. in a 50 ml sealed tube overnight. After completion of reaction (byTLC) ethanol was concentrated on rota vapor and excess of methyl aminewas removed by co evaporation with diethyl ether for three four times.Crude compound 602 thus obtained was used as such for the next reaction.Yield (1.20 gm, Crude). ¹H NMR (400 MHz, CDCl₃) δ=4.68 (d, 1H),4.58-0.46 (d, 1H), 4.47-4.5 (m, 1H), 3.43-3.47 (m, 1H), 3.39 (s, 3H),2.81-2.94 (m, 2H), 2.66 (s, 3H), 1.47 (s, 3H), 1.29 (s, 3H). LCMS [M⁺H]:218.3

Synthesis of Compound 603

To a stirred solution of 602 (0.38 g, 0.00175 moles) was dissolved in asolution of 10 ml (1:1) Dioxane: water and cooled to 0° C. To thismixture was added solid NaHCO₃ (0.367 gm, 0.00143752 moles) in portions.After 30 min of interval 50% solution of Cbz-Cl in toluene (0.448 g,0.002626 ml) was added slowly and resulting mixture was then stirred atroom temperature for 3 h. After completion of the reaction (by TLC),volatilities were removed under vacuum. The residue thus obtained wasdissolved in ethyl acetate (20 ml), washed with water (2×10 ml), driedover anhyd. Na₂SO₄ and concentrated under vacuum to give crude masswhich was purified by column chromatography on neutral alumina using 3%ethyl acetate in hexane as eluent. Yield (0.5 gm, 75%). ¹H NMR (400 MHz,CDCl₃) δ=7.24-7.35 (m, 5H), 5.24-5.04 (m, 2H), 4.94-4.91 (m, 1H),4.74-4.46 (m, 2H) 4.31 (br, 1H), 3.84-3.65 (m, 1H), 3.31 (s, 3H),3.16-3.02 (m, 1H), 2.98 (s, 3H), 1.46-1.43 (m, 3H), 1.29-1.20 (br., 3H).(note: NMR shows presence of rotamers). LCMS [M⁺H]: 352.4, [M⁺Na]:374.2.

Synthesis of Compound 604

Compound 603 (1.5 gm, 0.00426 moles) was dissolved in 30 ml of 70% aq.Acetic acid in a 100 ml single neck RBF equipped with reflux condenserand mixture was heated to reflux for 2 h. After completion of reaction(by TLC) acetic acid was removed under reduced pressure and residue wasdirectly subjected to column chromatography on 60-120 mesh silica gelusing 0-5% of methanol in dichloromethane as eluent to give purecompound 604 as sticky mass. Yield (0.510 gm, 39.23%). ¹H NMR (400 MHz,CDCl₃) δ=7.35 (m, 5H), 5.12-5.13 (m, 2H), 4.8 (m, 1H), 4.02-4.06 (m,3H), 3.46-3.60 (m, 2H), 3.29-3.46 (m, 4H), 3.04 (s, 3H), 2.58 (br, 1H).LCMS: [M⁺H]: 312.0, [M⁺NH₄]: 328.9

Synthesis of Compound 605

A single neck 100 ml RBF was charged with Compound 6 (0.5 gm, 0.00160moles), Dimethyl acetal of dilinoleyl ketone 7 (1.101 gm, 0.00192 mol)and 25 ml anhydrous toluene. To this catalytic amount of pTSA (5 mg) wasadded and reaction mixture was heated to reflux over soxhlet apparatusfor 5 h. Progress of the reaction was monitored by TLC. After completionof reaction, toluene was evaporated off and residue obtained waspurified by column chromatography on neutral alumina using 2% ethylacetate in hexane as eluent. Yield (0.600 g, 45%). ¹H NMR (400 MHz,CDCl₃) δ=7.35 (m, 5H), 5.37-5.30 (m, 8H), 5.20-5.08 (m, 2H), 4.94-4.91(m, 1H), 4.91-5.05 (m, 1H), 4.72-4.45 (m, 2H), 4.33-4.30 (m, 1H),3.77-3.62 (m, 1H), 3.30 (s, 3H), 3.17-3.08 (m, 1H) 2.98 (s, 3H),2.77-2.74 (m, 4H), 2.06-2.01 (m, 8H), 1.61-1.60 (m, 2H), 1.49 (m, 2H),1.35-1.26 (m, aliphatic protons), 0.89-0.85 (m, 6H). [M+H]=820 present.HPLC Purity:—95.78%.

Synthesis of ALNY-151

Prepared the compound ALNY-151 by following similar procedure ascompound ALNY-100, using N-Cbz-compound of the corresponding product(0.5 g, 0.6 mmol, 1.0 eq) and 1M solution of LAH in THF (0.9 mL, 0.9mmol, 1.5 eq), gave 0.32 g (76%) of the pure product ALNY-151. ¹H NMR(400 MHz, CDCl₃) δ 5.51-5.19 (m, 8H), 4.95 (s, 1H), 4.60 (dd, J=29.4,6.0, 2H), 4.25 (t, J=7.5, 1H), 3.33 (s, 3H), 2.77 (t, J=6.4, 4H), 2.38(dd, J=7.5, 5.2, 2H), 2.27 (s, 6H), 2.05 (q, J=6.7, 8H), 1.65 (dd,J=10.1, 5.9, 2H), 1.50 (d, J=8.9, 2H), 1.33 (ddd, J=18.9, 12.2, 7.1,36H), 0.89 (t, J=6.8, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 130.40, 130.36,128.17, 128.14, 116.25, 109.69, 85.68, 85.09, 83.54, 77.54, 77.23,76.91, 63.06, 54.95, 46.17, 37.20, 36.95, 31.75, 30.11, 30.05, 29.91,29.89, 29.78, 29.71, 29.58, 29.55, 29.53, 27.48, 27.46, 27.42, 25.85,24.38, 23.33, 22.80, 14.30. Calc. mass for the C₄₅H₈₁NO₄: 700.1; found700.5.

Example 19 Synthesis of ALNY-171

Synthesis of Compound 682

A procedure analogous to that described for the preparation of compound651 was followed to give compound 682 in 26% yield. ¹H NMR (400 MHz,CDCl₃) δ 7.61-7.14 (m, 5H), 6.13 (s, 1H), 5.29-5.01 (m, 2H), 3.97 (s,0.5H), 3.67 (s, 0.5H), 2.95 (d, J=19.5, 2H), 2.83 (d, J=29.7, 1H),1.71-1.52 (m, 2H), 1.52-1.38 (m, 1H), 1.31-1.08 (m, 2H); ¹³C NMR (101MHz, CDCl₃) δ 156.81, 138.71, 137.10, 137.01, 135.77, 128.39, 127.80,127.74, 127.67, 67.00, 66.72, 56.33, 46.77, 45.86, 40.72, 31.65, 29.75,29.54, 12.64, 12.10, 11.57; Electrospray MS (+ve): Molecular weight forC16H20NO2 (M+H)⁺ Calc. 258.1; Found 258.0.

Synthesis of Compound 683

Compound 683 (exo isomer) was successfully separated from exo/endomixture of compound 682 by preparative HPLC. The exo isomer wasconfirmed by NOE experiments. ¹H-NMR (400 MHz, CDCl₃) δ=7.35-7.28 (m,5H), 6.11 (s, 2H), 5.16-5.08 (m, 2H), 3.96 (b, 1H), 2.91 (s, 3H), 2.85(br, 1H), 2.78 (br, 1H), 1.46-1.24 (m, 4H). LCMS [M+H]: 258.2,[M+NH₄]:275.3, HPLC purity: 100%.

Synthesis of Compound 684

Compound 683 (1 g, 0.003891 mol) was dissolved in a mixture of 33 ml of(10:1) acetone and water in a 100 ml single neck RBF and to this wereadded NMO (1.36 g, 0.1167 mol) and 0.75 ml solution of 6.66% OsO₄ (0.05g, 0.00014 mol) in tert-butyl alcohol successively at room temperature.Progress of the reaction was monitored by TLC. After completion of thereaction (2 h) reaction mixture was quenched with aqueous Na₂SO₃solution (10 ml) and the mixture was stirred for 1.0 h. Aqueous phasewas then extracted with dichloromethane (2×50 ml). Organic phase waswashed once with sat. aq. NaHCO₃ (30 ml) solution followed by brine(1×25 ml). Organic layer was then dried over anhyd. Na₂SO₄ andconcentrated in vacuum. Crude material thus obtained was subjected to(60-120 mesh) silica gel column chromatography using 3% methanol indichloromethane as eluent which afforded the pure product as a thickliquid. Yield: 0.7 g (62%) ¹H NMR (400 MHz, CDCl₃) δ=7.33-7.28 (m, 5H),5.14-5.07 (dd, 2H), 3.87 (br, 1H), 3.76 (br, 1H), 3.72-3.69 (br, 1H),2.91 (m, 1H), 2.85 (s, 3H), 2.75 (br, 1H), 2.19 (m, 2H), 1.82-1.79 (m,1H), 1.68-1.66 (m, 1H), 1.44-1.40 (m, 2H). LC-MS: [M+H]-291.9 present.HPLC purity: 93% (chiral column) (Note: ¹H NMR and HPLC confirmed theformation of only one diastereomer)

Synthesis of 685

A mixture of compound 684 (0.7 g, 0.002405 mol), dilinoleyl ketone (1.9g, 0.003608 mol), 50 ml toluene and catalytic amount of p-TSA in a 100ml single neck RBF were stirred and heated to reflux vigorously overSoxhlet apparatus containing 4A molecular sieves and 50 ml toluene.Progress of the reaction was monitored by TLC. After completion of thereaction (˜8 h), mixture was cooled to room temperature and then toluenewas removed in vacuum to give crude mass which was purified by columnchromatography on neutral alumina using 1% ethyl acetate in hexane aseluting system to afford pure product 685 as pale yellow oil.

Yield: 1.5 g (78%) ¹H NMR (400 MHz, CDCl₃) δ=7.26-7.20 (m, 5H),5.29-5.19 (m, 8H), 5.06-4.97 (dd, 2H), 3.91-3.87 (m, 2H), 3.69 (m, 1H),2.77 (s, 3H), 2.66 (m, 4H), 2.21-2.18 (m, 2H), 1.97-1.92 (m, 8H),1.71-1.68 (m, 1H), 1.56-1.52 (m, 2H), 1.44 (m, 1H) 1.38 (m, 2H),1.30-1.17 (m, aliphatic protons), 0.78 (t, 6H). MS—[M+H]-800.1, HPLCpurity-90.91%.

Synthesis of ALNY-171

Prepared the compound ALNY-171 by following similar procedure ascompound ALNY-100, using N-Cbz-compound 685 (1.4 g, 1.75 mmol, 1.0 eq)and 1M solution of LAH in THF (2.62 mL, 2.62 mmol, 1.5 eq), gave 1.0 g(84%) of the pure product 17. ¹H NMR (400 MHz, CDCl₃) δ 5.50-5.20 (m,8H), 3.91 (dd, J=15.3, 5.4, 2H), 2.77 (t, J=6.3, 4H), 2.43 (s, 1H), 2.29(d, J=4.0, 1H), 2.18 (s, 6H), 2.04 (q, J=6.7, 8H), 1.64 (dd, J=10.4,5.7, 4H), 1.50 (s, 2H), 1.45-1.13 (m, 39H), 0.89 (t, J=6.7, 6H). ¹³C NMR(101 MHz, cdcl₃) δ 130.41, 130.39, 130.38, 130.34, 128.19, 128.15,113.17, 81.76, 81.21, 77.54, 77.22, 76.90, 65.76, 44.14, 43.40, 40.30,36.17, 35.25, 32.05, 31.75, 30.21, 29.89, 29.77, 29.74, 29.71, 29.57,29.52, 29.11, 27.46, 27.42, 25.85, 24.82, 24.08, 22.79, 14.29. Calc.mass for the C₄₆H₈₁NO₂: 680.1; found 680.5.

Example 20 Synthesis of ALNY-162, 163 and 164

Synthesis of Compound 691

Compound 690 (25 gm, 0.09615 moles) was dissolved in 250 ml dry THF in a500 ml two neck RBF and cooled to 0° C. To this solution was added PPh₃(25.2 gm, 0.09615 moles) followed by DEAD (15.79 ml, 0.09615 moles)under nitrogen atmosphere and the resulting mixture was stirred for 15min. Diphenyl phosphoryl azide (20.7 ml, 0.09615 moles) was then addedinto it drop wise at 0° C. Reaction mixture was then brought to roomtemperature slowly and stirred overnight. Upon completion of thereaction (by TLC) volatilities were removed under vacuum and crude masswas purified by silica gel (60-120 mesh) column chromatography using DCMas eluent to afford compound 691 as viscous liquid. Yield-19 gm (70.37%)1H NMR (CDCl3, 400 MHz): δ=5.54-5.53 (d, 1H J=5.0 Hz), 4.63-4.60 (dd, 1HJ=2.48 & 7.88 Hz), 4.33-4.31 (m, 1H), 4.19-4.17 (dd, 1H J=1.96 Hz & 7.92Hz), 3.92-3.88 (m, 1H), 3.52-3.47 (dd, 1H J=7.92 & 12.68 Hz), 3.37-3.32(dd, 1H J=5.4 & 12.72 Hz), 1.53 (s, 3H), 1.44 (s, 3H) 1.33 (s, 6H)

Synthesis of Compound 692

A 250 ml single neck RBF was charged with compound 691 (12 gm, 0.04195moles) and 120 ml Acetic acid:water (1:1). The resulting solution washeated at 75° C. for 4 h. Reaction was monitored by TLC. Upon completionof the reaction, mixture was concentrated under vacuum and crude solidthus obtained was washed with DCM (3×50 ml) to yield pure compound 692as an off white solid. Yield-6 gm 1H NMR (D2O, 400 MHz): δ=5.26-5.25 (d,1H J=3.76 Hz), 4.20-4.16 (m, 1H), 3.94 (m, 1H), 3.86-3.76 (m, 2H),3.54-3.43 (m, 2H)

Synthesis of Compound 693

Compound 692 (5.5 gm, 0.02669) was dissolved in (1:1) mixture ofmethanol: water (60 ml) in a 250 ml single neck RBF and degassed withnitrogen for 20 minutes. To this solution (20 wt %) Pd (OH)2/C (1.2 g,20 wt % of compound 3) was added at once under nitrogen. Reactionmixture was then flushed with hydrogen gas and stirred under balloonpressure of hydrogen gas for 3 days. (Note: on second and third day Igof (20 wt %) Pd (OH) 2/C was added into the reaction mixture and freshlyfilled hydrogen gas balloon was used for the reaction). After completionof the reaction, mixture was flushed with nitrogen and then filteredthrough celite bed. Filtrate and washings of (1:1) methanol and waterwere collectively evaporated under vacuum to yield crude compound 693 asoff white solid. Yield-4.5 gm 1H NMR (D2O, 400 MHz): δ=4.02-4.00 (d, 2H,J=6.3 Hz), 3.88-3.84 (m, 2H), 3.08-3.03 (dd, 2H J=4.32 & 14.76 Hz),2.88-2.83 (dd, 2H J=4.6 & 14.76 Hz).

Synthesis of 694

Compound 693 (4 gm, 0.0245 mol) and NaHCO₃ (5.14 gm, 0.06125 mol) weredissolved in (1:1) dioxan/water (30 ml) in a 100 ml single neck RBF andcooled to 0° C. After stirring the mixture for 10 minutes 50 wt % Cbz-Cl(8.25 g, 0.049 moles) in toluene (16.5 ml) was added slowly. Reactionmixture was then warmed to ambient temperature and monitored by TLC.After completion of the reaction (˜2 h by TLC) reaction mixture wasconcentrated under vacuum. Crude material obtained was purified bysilica gel (60-120 mesh) column chromatography using 0-20% methanol inDCM as eluent to afford the title compound Alny-13-001 as white solid.Yield-2.4 gm (33.33%) 1H NMR (400 MHz in MeOD): δ=7.38-7.25 (m, 5H),5.11-5.09 (d, 2H, J=5.84 Hz), 3.97-3.80 (m, 6H), 3.33-3.25 (m, 2H)LC-MS-[M+H]-298 present.

Synthesis of 695A and 695B

A 250 ml single neck flask equipped with a magnetic stir bar and aSoxhlet apparatus containing 4A molecular sieves and 50 ml toluene, wascharged with 694 (2.4 gm, 0.008081 mol), dilinoleyl ketone 7 (6.38 gm,0.01212 mol) and p-TSA (catalytic). To this mixture was added 30 mltoluene followed by methanol 3 ml. Resulting solution was stirred andheated to reflux vigorously for 3 h. Progress of the reaction wasmonitored by TLC. After complete disappearance of the starting 694 onTLC, toluene was evaporated and residue obtained was purified by columnchromatography on neutral alumina to get two different products as titlecompounds. 695A:—eluted with 12% ethyl acetate in hexane. Yield (900 mg)1H NMR (400 MHz IN CDCl3): δ=7.34-7.31 (m, 5H), 5.40-5.16 (m, 8H),5.15-5.14 (m, 2H), 4.13 (m, 1H), 4.03-3.98 (m, 4H), 3.66-3.62 (m, 1H),3.21 (s, 1H), 3.14-3.10 (d, 1H, J=15.44 Hz), 2.77-2.74 (t, 4H),2.26-2.22 (m, 1H), 2.06-2.01 (m, 8H), 1.63-1.59 (m, 4H), 1.35-1.26 (m,aliphatic protons from linoleyl group), 0.89-0.85 (m, 6H)LC-MS—[M+H]-806.30 HPLC—95.04%. 695B:—eluted with 9% ethyl acetate inhexane. Yield (500 mg) 1H NMR (400 MHz IN CDCl3): δ=7.38-7.25 (m, 5H),5.40-5.28 (m, 8H), 5.16-5.12 (m, 2H), 4.43-4.36 (m, 2H), 4.12-4.11 (m,1H), 4.03-3.99 (m, 3H), 3.62 (br s, 1H), 3.35-3.28 (m, 2H), 1.95 (br s,1H), 2.77-2.74 (m, 4H), 2.06-2.01 (m, 8H), 1.58 (m, 4H), 1.35-1.26 (m,aliphatic protons from linoleyl group), 0.89-0.84 (m, 6H)LC-MS—[M+H]-806.40 HPLC—95.12%.

Synthesis of ALNY-164 and ALNY-164B

Prepared the compound ALNY-164 and ALNY-164B by following the similarprocedure used for ALNY-100, using N-Cbz-compound 695A and 695B.

Example 21 Synthesis of ALNY-163 and ALNY-163B

Synthesis of Compound 697

Compound 692 (9.5 gm, 0.04611 μmol) was dissolved in 30 ml of benzylalcohol in a 500 ml two neck RBF at 80° C. To this solution was addedBF3.Et2O (6.54 gm, 0.04611 mol) slowly under nitrogen atmosphere.Reaction was continued for 1 h. Progress of the reaction was monitoredby TLC. Upon completion of the reaction (˜2 h) reaction mixture wasdirectly chromatographed on 60-120 mesh silica gels. Benzyl alcohol waseluted with 20% EtOAc in hexane at first and pure compound was theneluted with ethyl acetate. Yield: (8.2 gm, 60.29%) 1H NMR (MeOD, 400MHz): δ=7.40-7.21 (m, 5H), 4.77-4.56 (m, 2H), 4.09-4.08 (m, 1H),3.98-3.95 (m, 1H), 3.80-3.77 (m, 2H), 3.72-3.68 (m, 1H), 3.57-3.51 (m,1H), 3.27-3.23 (m, 1H)

Synthesis of Compound 698

To a stirred solution of 697 (8.2 gm, 0.0276 mol) in 80 ml anhydrous DMFcontained in a 250 ml two neck RBF, was added PTSA (catalytic) and 2,2-DMP (22 ml, Excess) under nitrogen atmosphere. Resulting mixture wasstirred overnight at room temperature Reaction was monitored by TLC.After completion of the reaction, mixture was diluted with water (250ml) was extracted well with diethyl ether (100 ml×3 ml). Organic layerwas separated, dried over sodium sulphate and concentrated under vacuumto yield crude compound which was purified by (230-400 mesh) silica gelcolumn chromatography using EtOAc in hexane as eluent. Fraction I waseluted with 12% ethyl acetate in hexane-Yield: (5 gm, 53.19%). 1H NMR(CDCl3, 400 MHz): δ=7.38-7.27 (m, 5H), 4.97-4.96 (d, 1H J=3.88 Hz),4.86-4.83 (d, 1H J=11.56 hz), 4.62-4.59 (d, 1H J=11.64 Hz), 4.32-4.29(m, 1H), 4.21-4.10 (m, 2H), 3.90-3.86 (m, 1H), 3.59-3.54 (dd, 1H J=8.48& 12.84 Hz), 3.33-3.28 (dd, 1H J=4.36 & 12.88 Hz), 2.42-2.40 (d, 1HJ=5.8 Hz) 1.58 (s, 3H), 1.48 (s, 3H). [Note: Fraction II was eluted with20% ethyl acetate in hexane-yield: (1.4 gm, 15%,

Synthesis of Compound 699

To a stirred solution of compound 698 (5 gm, 0.01483 mol) in 50 ml DryDMF in a 250 ml two neck RBF was added (60% suspension in mineral oil)sodium hydride (1.42 gm, 0.0296 mol) in portions for a period of 10minute at 0° C. under nitrogen atmosphere. After stirring the mixturefor 15 min at 0° C., MeI (1.1 ml, 0.0179 mol) was added slowly andreaction mixture was stirred additional for 1 h. Reaction was monitoredby TLC. After completion, reaction mixture was quenched with crushed ice(˜200 g) and aqueous phase was extracted with EtOAc (100 ml×3). Organiclayer was then washed with water (100 ml×2) to remove traces of DMF,dried over Na2SO4, and then concentrated on rotary evaporator to get thetitle compound 699 as oily compound which was used directly withoutfurther purification. Yield (4.5 gm, 86.04%) 1H NMR (CDCl3, 400 MHz):δ=7.40-7.29 (m, 5H), 5.00-4.99 (d, 1H J=3.52 Hz), 4.77-4.74 (d, 1HJ=12.16 Hz), 4.61-4.58 (d, 1H J=12.16 Hz), 4.31-4.28 (dd, 1H J=5.48 &7.64 Hz), 4.15-4.11 (m, 2H), 3.60-3.55 (dd, 1H J=8.08 & 12.8 Hz), 3.42(s, 3H), 3.38-3.34 (m, 2H), 1.51 (s, 3H), 1.32 (s, 3H).

Synthesis of Compound 700

A 100 ml single neck RBF was charged with compound 699 (4.4 gm, 0.12mol), 44 ml of mixture of AcOH:water (8:2), and mixture was heated to80° C. for 4 h. Reaction was monitored by TLC. After completion of thereaction, the mixture was extracted with DCM (3×40 ml). Organic layerwas washed with water (2×25 ml) followed by saturated aq. NaHCO3solution (2×30 ml). Organic layer was then dried over Na2SO4 and solventwas evaporated off in vacuum to yield compound 700 as white solid. Yield(3.7 gm, 95.60%) 1H NMR (MeOD, 400 MHz): δ=7.41-7.27 (m, 5H), 5.08-5.07(d, 1H J=3.7 Hz), 4.77-4.74 (d, 1H J=11.88 Hz), 4.59-4.56 (d, 1H,J=11.88 Hz), 3.97-3.94 (dd, 1H J=4.0 & 8.44 Hz), 3.85-3.79 (m, 2H),3.58-3.52 (m, 1H), 3.50-3.47 (m, 1H), 3.37 (s, 3H), 3.27-3.22 (m, 1H).13C NMR (MeOD, 100 MHz) δ=137.24, 128.03, 127.99, 127.52, 94.84, 77.75,70.02, 69.93, 68.82, 68, 73, 56.91, 51.24.

Synthesis of Compound 701

Compound 700 (3 gm, 0.0097 mol) was dissolved in (30 ml) methanol in a250 ml single neck RBF and degassed with nitrogen for 20 minutes. Tothis solution (20 wt %) Pd (OH)2/C (0.8 gm, ˜20 wt % of compound 7) wasadded at once under nitrogen. Reaction mixture was then flushed withhydrogen gas and stirred under balloon pressure of hydrogen gas for 3days. (Note:on second and third day 0.8 g of (20 wt %) Pd (OH) 2/C wasadded into the reaction mixture and freshly filled hydrogen gas balloonwas used for the reaction). After completion of the reaction, mixturewas flushed with nitrogen and then filtered through celite bed. Filtrateand washings of (1:1) methanol and water were collectively evaporatedunder vacuum to yield crude compound 701 as sticky mass. Yield (1.8 gm,Crude) 1H NMR (D2O, 400 MHz): δ=4.13-4.11 (m, 1H), 3.96-3.94 (m, 1H),3.84-3.80 (m, 1H), 3.44-3.43 (m, 1H), 3.33 (s, 3H), 3.03-2.98 (m, 2H),2.95-2.90 (m, 1H), 2.86-2.81 (m, 1H). 13C NMR (D2O, 100 MHz): δ=82.84,75.60, 73.79, 72.09, 57.78, 53.06, 49.98

Synthesis of Compound 702

Compound 701 (1.8 g, 0.0101 mol) and NaHCO3 (2.121 g, 0.02525 mol) weredissolved in (1:1) dioxan and water (50 ml) in a 100 ml single neck RBFand cooled to 0° C. After stirring the mixture for 10 minutes 50 wt %Cbz-Cl (6.9 ml, 0.0203 mol) in toluene was added slowly. Reactionmixture was then warmed to ambient temperature and monitored by TLC.After completion of the reaction (˜2 h by TLC) reaction mixture wasconcentrated under vacuum. Residual part was diluted with water (20 ml)and extracted with DCM (3×30 ml) Organic layer was then separated, driedover anhydrous Na2SO4 and concentrated under vacuum to yield the Crudematerial which was purified by silica gel (60-120 mesh) columnchromatography using 0-20% methanol in DCM as eluent to afford the titlecompound 702 as off white solid. Yield (0.9 gm, 29%) 1H NMR (MeOD, 400MHz): δ=7.37-7.28 (m, 5H), 5.15-5.06 (m, 2H), 4.01-4.00 (m, 1H),3.89-3.88 (m, 1H), 3.84-3.83 (m, 1H), 3.75-3.69 (m, 2H), 3.61-3.60 (m,1H), 3.55-3.30 (m, 5H). (note: exists as rotamers). LC-MS [M+H]-312.30

Ketalization Products: 703A & 703B

A 100 ml single neck flask equipped with a magnetic stir bar and aSoxhlet apparatus containing 4A molecular sieves and 50 ml toluene, wascharged with 702 (0.9 gm, 0.00289 mol), dilinoleyl ketone (3.04 gm,0.005787 mol) and p-TSA (catalytic). To this mixture was added 30 mltoluene followed by methanol 3 ml. Resulting solution was stirred andheated to reflux vigorously for 3 h. Progress of the reaction wasmonitored by TLC. After complete disappearance of the starting 702 onTLC, toluene was evaporated off and residue obtained was purified bycolumn chromatography on neutral alumina to get two different productsas title compounds.

703A was eluted with 7-9% ethyl acetate in hexane system.

Yield: (0.500 gm) 1H NMR (CDCl3, 400 MHz): δ=7.38-7.28 (m, 5H),5.40-5.28 (m, 8H), 5.20-5.15 (m, 2H), 4.42-4.33 (m, 2H), 4.23 (m, 1H),4.00 (m, 1H), 3.88-3.87 (m, 1H), 3.73 (m, 1H), 3.61-3.59 (m, 1H), 3.50(s, 3H), 3.40-3.20 (m, 3H), 2.77-2.74 (m, 4H), 2.06-2.01 (m, 8H),1.68-1.56 (m, 2H), 1.35-1.29 (m, aliphatic protons), 0.89-0.85 (m, 6H)MASS [M+H]-820.6, HPLC (ELSD) purity-95.69%

703B was eluted with 12-15% ethyl acetate in hexane system.

Yield: (0.400 g) 1H NMR (CDCl3, 400 MHz): δ=7.36-7.30 (m, 5H), 5.38-5.30(m, 8H), 5.15-5.13 (m, 2H), 4.21-4.10 (m, 3H), 4.09-4.00 (m, 1H),3.91-3.80 (m, 2H), 3.47 (s, 3H), 3.39 (m, 1H), 3.30-3.10 (m, 3H),2.77-2.74 (m, 4H), 2.36-2.33 (d, 1H, J=13.2 Hz), 2.06-2.01 (m, 8H),1.63-1.59 (m, 2H), 1.37-1.23 (m, aliphatic protons), 0.89-0.86 (m, 6H)MASS [M+H]-820.5, HPLC (ELSD) purity-95.12%.

Synthesis of ALNY-163 and ALNY-163B

Prepared the compound ALNY-163 and ALNY-163B by following the similarprocedure used for ALNY-100, using N-Cbz-compound 703A and 703B.

Example 22 Synthesis of ALNY-205, ALNY-209 and ALNY-209B

Synthesis of Compound 706

Benzyl mannopyranoside 705 (5 g, 0.0185 mol) was dissolved in drypyridine (50 ml) in a 250 ml two neck RBF and cooled to 0° C. To thisstirred was added slowly a 25 ml solution of p-tosyl chloride (3.7 g,0.0194 mol) in anhydrous DCM under nitrogen atmosphere. Resultingmixture was allowed to warm to room temperature and progress of thereaction was monitored by TLC. After completion of the reaction (˜4 h.by TLC), reaction mixture was diluted with DCM (250 ml) and washed with1N HCl (2×100 ml) followed by saturated NaHCO3 (2×50 ml) and brine (50ml) respectively. Organic layer was then separated, dried over anhydrousNa2SO4, filtered and then concentrated under vacuum to give crudematerial which was subjected to silica gel (60-120 mesh) columnchromatography and eluted with ethyl acetate to afford pure compound 706as off white solid. Yield: 4.9 g (62.5%) 1H NMR (DMSO, 400 MHz): δppm=7.80-7.76 (d, 2H J=8.24 Hz), 7.48-7.46 (d, 2H J=8.16 Hz), 7.43-7.29(m, 5H), 5.06-5.04 (d, 1H J=5.8 Hz), 4.93-4.92 (d, 1H, J=4.24 Hz),4.78-4.76 (d, 1H J=6 Hz), 4.65 (d, 1H J=0.72 Hz), 4.55-4.52 (d, 1HJ=11.8 Hz), 4.38-4.35 (d, 1H J=11.84 Hz), 4.23-4.05 (dd, 1H), 3.61 (m,1H), 3.54 (m, 1H), 3.45-3.42 (m, 1H), 2.40 (s, 3H)

Synthesis of Compound 707

A 500 ml single neck RBF equipped with a magnetic bar and refluxcondenser was charged with compound 706 (9.7 g, 0.02287 mol), sodiumazide (10.4 g, 0.1600 mol), ammonium chloride (6.12 g, 0.1143 mol) and(9:1) Ethanol: water (150 ml). Resulting mixture was stirred and heatedto reflux for 24 h. Upon completion of the reaction (monitored by TLC)solvent was removed on rotary evaporator and crude mass waschromatographed on silica gel (100-200 mesh) using ethyl acetate to givecompound 707 as white solid. Yield: 4 g (59.2%) 1H NMR (DMSO, 400 MHz):δ ppm=7.43-7.28 (m, 5H), 5.01-5.00 (d, 1H, J=5.88 Hz), 4.93-4.92 (d, 1H,J=4.24 Hz), 4.74-4.72 (m, 2H), 4.68-4.65 (d, 1H J=11.88 Hz), 4.49-4.46(d, 1H J=11.88 Hz), 3.65-3.64 (m, 1H), 3.60-3.54 (m, 1H), 3.52-3.47 (m,1H), 3.46-3.38 (m, 3H).

Synthesis of Compound 708

Compound 707 (4 g, 0.01355 mol) was dissolved in (3:1) mixture ofmethanol and water (60 ml) in a 250 ml single neck RBF and degassed withnitrogen for 20 minutes. To this solution (20 wt %) Pd (OH)2/C (1 g, 25wt % of compound 3) was added at once under nitrogen. Reaction mixturewas then flushed with hydrogen gas and stirred under balloon pressure ofhydrogen gas for 3 days. (Note: on second and third day 0.5 g of (20 wt%) Pd (OH) 2/C was added into the reaction mixture and freshly filledhydrogen gas balloon was used for the reaction). After completion of thereaction, mixture was flushed with nitrogen and then filtered throughcelite bed. Filtrate and washings of (3:1) methanol and water werecollectively evaporated under vacuum to yield crude compound 708 as offwhite solid. Yield: 2 g (90%). 1H NMR (D2O, 400 MHz): δ ppm=4.09-4.07(m, 2H), 3.91-3.89 (m, 2H), 2.98-2.86 (m, 4H).

Synthesis of 709

Compound 708 (2 g, 0.01225 mol) and NaHCO3 (2.57 g, 0.0306 mol) weredissolved in (1:1) dioxan:water (40 ml) in a 100 ml single neck RBF andcooled to 0° C. After stirring the mixture for 10 minutes 50 wt % Cbz-Cl(3.13 g, 0.01838 mol) in toluene (6.26 ml) was added slowly. Reactionmixture was then warmed to ambient temperature and monitored by TLC.After completion of the reaction (˜3 h by TLC) reaction mixture wasconcentrated under vacuum. Residue thus obtained was stirred with 30 mlof 10% methanol in DCM solution and filtered. Filtrate was evaporatedoff and crude material obtained was purified by silica gel (60-120 mesh)column chromatography using 10% methanol in DCM as eluent to afford thetitle compound 709. Yield: 2 g (55%). 1H NMR (MeOD, 400 MHz): δppm=7.38-7.21 (m, 5H), 5.12 (s, 2H), 4.10-4.04 (m, 2H), 3.91 (s, 2H),3.58-3.52 (m, 4H).

Ketalization: Synthesis of 710A, 710B and 710C

A 250 ml single neck flask equipped with a magnetic stir bar and aSoxhlet apparatus containing 4A molecular sieves and 50 ml toluene, wascharged with 709 (2 g, 0.00673 mol), dilinoleyl ketone (10.64 g, 0.0204mol) and p-TSA (0.129 g, 0.00067 mol). To this mixture was added 90 mltoluene followed by methanol 10 ml. Resulting solution was stirred andheated to reflux vigorously for 3 h. Progress of the reaction wasmonitored by TLC. After complete disappearance of the starting 709 onTLC, reaction was stopped, toluene was evaporated off and residueobtained was purified by column chromatography on neutral alumina to getthree different products as title compounds.

710A: eluted with 1.5-2% ethyl acetate in hexane-1.5 g 1H NMR (CDCl3,400 MHz): δ ppm=7.35-7.30 (m, 5H), 5.40-5.29 (m, 16H), 5.13-5.12 (m,2H), 4.36-4.16 (m, 6H), 2.92-2.74 (m, 10H), 2.06-2.01 (m, 16H),1.66-1.64 (m, 4H), 1.58-1.55 (m, 4H), 1.37-1.25 (m, aliphatic protons)0.89-0.86 (m, 12H).

710B: eluted with 25-30% ethyl acetate in hexane—1 g 1H NMR (CDCl3, 400MHz): ppm=7.36-7.30 (m, 5H), 5.38-5.30 (m, 8H), 5.14 (m, 2H), 4.35 (m,2H), 4.13-4.11 (m, 2H), 3.85-3.83 (m, 1H), 3.72-3.71 (m, 1H), 3.55-3.54(m, 1H), 3.33 (m, 1H), 2.77-2.74 (m, 4H), 2.46 (s, 1H) 2.36 (s, 1H),2.06-2.01 (m, 8H), 1.63-1.61 (m, 4H), 1.37-1.25 (m, aliphatic protons)0.89-0.85 (m, 6H). Mass [M+H]-806.30 present HPLC purity-94.85%

710C: eluted with 5% methanol in DCM—0.52 g. 1H NMR (CDCl3, 400 MHz): δppm=7.35-7.30 (m, 5H), 5.40-5.31 (m, 8H), 5.17-5.14 (m, 2H), 4.60 (m,1H), 4.41-4.37 (m, 2H) 4.33-4.29 (m, 2H), 4.13 (m, 1H), 3.85-3.81 (m,1H), 3.05-3.01 (m, 1H), 2.77-2.76 (m, 4H), 2.56 (m, 1H), 2.36-2.34 (m,1H), 2.20 (m, 1H), 2.04-2.02 (m, 8H), 1.62-1.61 (m, 4H), 1.37-1.26 (m,aliphatic protons) 0.89-0.85 (m, 6H). Mass [M+H]-806.40 present HPLCpurity-91.52%

Synthesis of ALNY-205 and ALNY-209 and ALNY-209B

Prepared the compound ALNY-205, ALNY-209 and ALNY-209B by following thesimilar procedure used for ALNY-100, using N-Cbz-compound 710A, 710B and710C.

Synthesis of ALNY-148

Preparation of Compound 721: To a solution of dihydroxy-Cbz compound 720(0.8 g, 3.0 mmol, 1.0 eq), dilinoleyl ketone 7 (1.58 g, 3.0 mmol, 1.0eq) in toluene was added the PTSA (0.05 g, 0.3 mmol, 0.1 eq) andrefluxed under Dean-Stock apparatus until there is no starting materialleft. Cooled the reaction mixture, evaporated, directly loaded on columnchromatography and purified using hexane: ether (10%) as gradients toget 1.45 g of the pure compound 721 in 63% yields. ¹H NMR (400 MHz,CDCl₃) δ 7.46-7.26 (m, 6H), 5.49-5.21 (m, 8H), 5.13 (s, 2H), 3.75 (s,4H), 3.41 (t, J=10.4, 2H), 2.77 (t, J=6.4, 4H), 2.05 (q, J=6.7, 8H),1.69 (s, 2H), 1.65-1.48 (m, 6H), 1.42-1.16 (m, 37H), 0.89 (t, J=6.8,6H). Calc. mass for the C₅₁H₈₃NO₄: 773.6; found 796.5 (+Na).

Preparation of ALNY-148: Exactly repeated the experimental procedure asof the compound alny-100 using N-Cbz-compound 721 (1.44 g, 1.86 mmol,1.0 eq), 1M LAH in THF (3.72 mL, 3.72 mmol, 2.0 eq), which gave 1.04 g(86%) of the pure product. ¹H NMR (400 MHz, CDCl₃) δ 5.51-5.16 (m, 8H),3.75 (s, 2H), 2.77 (t, J=6.4, 4H), 2.55 (s, 2H), 2.33 (s, 1H), 2.28 (s,3H), 2.04 (q, J=6.8, 8H), 1.88-1.74 (m, 2H), 1.74-1.63 (m, 2H), 1.57(dd, J=10.2, 5.4, 4H), 1.45-1.15 (m, 37H), 0.88 (t, J=6.8, 6H). ¹³C NMR(101 MHz, CDCl₃) δ 130.40, 130.36, 128.15, 128.13, 112.67, 78.12, 77.55,77.23, 76.91, 73.59, 53.33, 46.31, 37.93, 36.66, 31.74, 30.17, 29.90,29.79, 29.73, 29.57, 29.53, 27.45, 27.41, 25.84, 24.32, 22.80, 14.31.Calc. mass for the C44H79NO2: 653.6; found 654.5.

Example 23 Synthesis of Ketal 530

Preparation of Compound 528

An ice-cold solution of compound 527 (10 g) in CHCl₃ (500 mL) is treatedall at once with m-chloroperbenzoic acid (1.1 eq), then warmed to r.t.and stirred for 18 h. Aqueous workup then column chromatography givescompound 528.

Preparation of 25 for ketone 7 and using a procedure analogous to thatdescribed for the synthesis of compound 505, compound 529 is obtained asa colorless oil.

Preparation of Compound 530

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 530 is obtained as a colorless oil.

Example 24 Synthesis of Ketal 534

Preparation of Compound 532

Using a procedure analogous to that described for the synthesis ofcompound 504, compound 532 is obtained as a colorless oil.

Preparation of Compound 533

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 533 (%) is obtained as a colorless oil.

Preparation of Compound 534

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 534 is obtained as a colorless oil.

Example 25 Synthesis of Ketal 538

Preparation of Compound 536

Using a procedure analogous to that described for the synthesis ofcompound 504, compound 536 is obtained as a colorless oil.

Preparation of Compound 537

Substituting ketone 27 for ketone 7, and using a procedure analogous tothat described for the synthesis of compound 505, compound 537 isobtained as a colorless oil.

Preparation of Compound 538

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 538 is obtained as a colorless oil.

Example 26 Synthesis of Ketone 12

The cyclic ketone 12 is synthesized using a similar procedure to thatreported in J. Org. Chem. 2005, 70, 5420-5425.

Example 27 Synthesis of Ketal 540

Preparation of Compound 539

Substituting ketone 12 for ketone 7, and using a procedure analogous tothat described for the synthesis of compound 505, compound 539 isobtained as a colorless oil.

Preparation of Compound 540

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 540 is obtained as a colorless oil.

Example 28 Synthesis of Ketone 15

The ketone 15 is synthesized starting from the dibromo ketal 13 via theGrignard reagent 14 as shown in good yields.

Example 29 Synthesis of Ketal 542

Preparation of Compound 541

Substituting ketone 15 for ketone 12, and using a procedure analogous tothat described for the synthesis of compound 505, compound 541 isobtained as a colorless oil.

Preparation of Compound 542

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 542 is obtained as a colorless oil.

Example 30 Synthesis of Ketone 18

Using a similar procedure to that used for the synthesis of ketone 15,starting from dibromide 16 the six membered cyclic ketone 18 issynthesized as shown.

Example 31 Synthesis of Ketal 544

Preparation of Compound 543

Substituting ketone 18 for ketone 12, and using a procedure analogous tothat described for the synthesis of compound 505, compound 541 isobtained as a colorless oil.

Preparation of Compound 544

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 544 is obtained as a colorless oil.

Example 32 Preparation of Compounds 545-550

The six diastereomers of 1,2-dihydroxy-4,5-diaminocyclohexane (compounds545-550) are prepared according to the procedure described in Journal ofMedicinal Chemistry, 30(8), 1327-36; 1987. Compounds 545 and 548 areobtained as racemates, compounds 546, 547, 549 and 550 are mesocompounds.

Example 33 Preparation of Compound 553

Preparation of Compound 551

Using procedures which are analogous to those described for thesynthesis of compounds 504 and 506, compound 551 is obtained as acolorless oil.

Preparation of Compound 552

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 552 is obtained as a colorless oil.

Preparation of Compound 553

Using a procedure analogous to that described for the synthesis ofcompound 506, compound 553 is obtained as a colorless oil.

Example 34 Preparation of Compound 556

Preparation of Compound 556

Using procedures which are analogous to those described for thesynthesis of compounds 551 to 506, compound 556 is obtained as acolorless oil.

Example 35 Synthesis of Optically pure Cyclic diol 25

The optically pure diol 25 is synthesized as shown above starting fromcommercially available (+)-tartaric acid. Treatment of tartaric acidwith benzylamine in refluxing xylene provided the imide 19 which onreduction with lithium aluminumhydride provided the pyrrolidine 20 inwhich the diol is protected with TBDMS and followed by alkylation withthe bromide 23 to give product 24 which is deprotected to isolate thepure diol 25 in good yields.

Example 36 Synthesis of Ketal 26

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 26 is obtained as a colorless oil.

Example 37 Synthesis of Ketal 30

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 30 is obtained as a colorless oil.

Example 38 Synthesis of Compound 406

Preparation of 401

Tosylate (250 g, 0.873 mol) was charged in a 7.5 L auto clave followedby methanolic ammonia (60%) (3750 mL) at −20° C. The pressure vessel wasclosed and stirred while allowing the reaction mixture to warm to roomtemperature. It was then heated to 90° C. and continued for 5 hrs at 90°C. (TLC). Reaction mixture was cooled to room temperature and theinternal pressure was released. It was then purged with nitrogen for 15minutes to remove excess of ammonia, evaporated at reduced pressure at30° C. to obtain crude residue. It was washed with diethyl ether (2×250ml) to remove impurities and dried at reduced pressure at 30-34° C. toafford the desired product along with the by-product sulfonamide. Thismixture has been taken as such for the next step (Yield 112 g, 95%). ¹HNMR (400 MHz, DMSO-d₆): δ 1.29 (S, 3H), 1.38 (S, 3H), 2.83 (m, 1H), 3.02(dd, 1H), 3.70 (m, 1H), 4.04 (t, 1H), 4.24 (m, 1H).

Preparation of 402

To a solution of 401 (112 g, 0.850 mol) in dichloromethane (560 mL) at0° C., was added 2,6 Lutidine (182.2 g, 1.70 mol) drop-wise during 15minutes. Then Cbz-Cl (147.50, 0.860 mol) was added drop-wise during 25minutes at same temperature. It was then stirred for another 15 minutesat 0° C. (TLC). It was diluted with dichloromethane (300 ml), washedwith 10% citric acid (3×250 ml) and brine (1×250 ml). The organic layerwas separated, dried over sodium sulfate and evaporated at reducedpressure at 45° C. to obtain crude product, which was purified by silicagel chromatography using ethyl acetate/hexane as eluent to afford pureproduct (Yield 174, 77%). ¹H NMR (400 MHz, CDCl₃): 1.33 (S, 3H), 1.40(S, 3H), 3.23 (m, 1H), 3.43 (m, 1H), 3.64 (t, 1H), 4.01 (t, 1H), 4.2 (m,1H), 5.60 (s, 2H), 5.24 (s, 1H), 7.31 (s, 5H).

Preparation of 403

To a solution of 402 (149 g, 0.5622 mol) in methanol and water (1:85,800 mL) was added PTSA (214 g, 2 eq.) at room temperature and stirredfor 15 minutes (TLC). It was then evaporated at reduced pressure toremove methanol and diluted with water (150 ml). Then pH of the aqueoussolution was adjusted to 8.5-9 using solid sodium carbonate andextracted several times with ethyl acetate to ensure complete recoveryof the product. The combined organic layer was dried over sodium sulfateand evaporated at reduced pressure to afford pure product (Yield, 118 g,90%) as white solid. ¹H NMR (CDCl₃, 400 MHz): δ 3.1 (s, 1H), 3.3 (m,3H), 3.6 (m, 2H), 3.8 (s, 1H), 5.1 (s, 2H), 5.3 (s, 1H), 7.3 (m, 5H).

Preparation of 405

To stirred mixture of 403 (170 g, 0.755 mol) and ketone 7 (199 g, 0.377mol) in anhydrous toluene (2 L), was added half of the quantity of PTSA(7.1 g, 0.1 eq) and heated to reflux at 130° C. bath using dean starkapparatus. After 6 hours second half of the quantity of PTSA (7.1 g, 0.1eq.) was added and reaction was continued for another 12 hours (TLC).Reaction mass was allowed to cool to room temperature and filtered toremove precipitated solids. The filtrate was washed with saturatedsodium bicarbonate solution, dried over sodium sulfate and evaporated atreduced pressure to obtain crude product, which was purified by silicagel chromatography using 5% ethyl acetate/hexane as eluent to affordpure product as color less liquid (153 g, 55%). ¹H NMR (CDCl₃): δ value:0.9 (t, 6H), 1.3 (m, 38H), 1.5 (m, 2H), 1.6 (s, 4H), 2.0 (m, 8H), 2.8(t, 4H), 3.2 (m, 1H), 3.5 (m, 1H), 3.6 (t, 1H), 4.0 (t, 1H), 4.2 (s,1H), 5.1 (s, 1H), 5.2 (s, 2H), 5.5 (m, 8H), 7.3 (m, 5H).

Preparation of 406

To LAH (16.6 g, 0.438 mol) under N₂ at 0° C. was added dry THF (1.3 L)and stirred for 30 minutes. To the suspension was added a solution of405 (161 g, 0.219 mol) in THF (300 ml) slowly by maintaining thetemperature at 0° C. After addition, the reaction mass was warmed to 40°C. During this period the inside temperature rose from 40 to 54° C. andafter few minutes again decreased to 40° C. Reaction was continued foranother 1 hr at 40° C. (TLC). It was then cooled to 0° C. and quenchedwith 100 ml of sat. sodium sulfate solution and diluted with ethylacetate (1 L). The solid formed was filtered through a pad of celitebed. The residue was washed several times with ethyl acetate. Thecombined filtrate was evaporated at reduced pressure to obtain crudeproduct, which was purified by silica gel chromatography usingdichloromethane/15% ethyl acetate as eluent to obtain pure product as anyellow liquid (99 g, 74%). ¹H NMR (CDCl₃): δ value: 0.96 (t, 6H), 1.3(m, 38H), 1.6 (m, 4H), 2.1 (q, 8H), 2.53 (s, 3H), 2.76 (dd, 2H), 2.82(q, 4H), 3.6 (t, 1H), 4.1 (t, 1H), 4.3 (m, 1H), 5.4 (m, 8H).

Example 39 Preparation of Compound 408

Compound 406 is treated with 406a under reductive amination conditionsas shown above to get 407. It is then treated with Sodium hydroxidesolution in Methanol/THF/Water to get 408.

Example 40 Preparation of Compound 410

Compound 406 is alkylated with methyl bromo acetate using potassiumcarbonate in THF/Water as shown above to get 409. It is then treatedwith Sodium hydroxide solution in Methanol/THF/Water to get 410.

Example 41

Compound 411 is prepared from the acetonide 411a using transketalysation. LAH reduction gives compound 412. It is then convert tothe diester 414 by reductive amination. Deprotection of 414 using NaOHgives compound 415.

Example 42 Preparation of Compound 418

Lipid acid 410 is treated with GalNAc amine 416 under peptide couplingconditions to get 417. Deprotect the acetate groups using LiOH givescompound 418.

Example 43 Preparation of Compound 421

Lipid acid 410 is treated with Mannose amine 419 under peptide couplingconditions to get 420. Deprotect the benzoate groups using LiOH givescompound 421.

Example 44 Preparation of Compound 424

Lipid acid 408 is treated with triantineary GalNAc amine 422 underpeptide coupling conditions to get 423. Deprotect the acetate groupsusing methyl amine at room temperature gives compound 424.

Example 45 Preparation of Compound 427

Lipid acid 408 is treated with triantineary mannose amine 425 underpeptide coupling conditions to get 426. Deprotection of benzoate groupsusing methyl amine at room temperature gives compound 427.

Example 46 Preparation of Compound 430

Lipid diacid 415 is treated with GalNAc amine 416 under peptide couplingconditions to get 429. Deprotection of acetate groups using methyl amineat room temperature gives compound 430.

Example 47 Preparation of Compound 432

Lipid diacid 415 is treated with mannose amine 419 under peptidecoupling conditions to get 431. Deprotection of acetate groups usingmethyl amine at room temperature gives compound 432.

Example 48 Preparation of Compound 438

Compound 436 and 437 react to give compound 438 under click chemistryconditions.

Example 49 Preparation of Compound 442

Preparation of 439

Compound 439 is prepared according to the procedure described in Dills,William L., Jr.; Covey, Thomas R.; Singer, Paul; Neal, Sharon;Rappaport, Mark S. “2,5-Anhydro-1-deoxy-D-lyxitol,2,5-anhydro-1-deoxy-D-mannitol, and 2,5-anhydro-1-deoxy-D-talitol.Synthesis and enzymic studies” Carbohydrate Research (1982), 99(1),23-31.

Preparation of 440

To a mixture of 439 (10 g, 67.53 mmol) and ketone 404 (18 g, 34 mmol) inanhydrous toluene (200 mL), is added PTSA (1.17 g, 0.2 eq) and heated toreflux at 130° C. bath using dean stark apparatus for 24 hr. Cool thereaction mixture to room temperature and wash with sodium bicarbonatesolution. Crude product is purified by chromatography to get the product440.

Preparation of 441

Compound 440 is treated with Methane sulfonylchloride in presence oftriethylamine in dichloromethane to get 441.

Preparation of 442

To a solution of 441 in EtOH, Dimethylamine in EtOH is added and heatthe reaction mixture in a pressure reactor at 70° C. for overnight toget 442.

Example 50 Preparation of Compound 445

Preparation of 443

Compound 443 is prepared according to the procedure described inBouchez, Veronique; Stasik, Imane; Beaupere, Daniel; Uzan, Raoul;“Regioselective halogenation of pentono-1,4-lactones. Efficientsynthesis of 5-chloro- and 5-bromo-5-deoxy derivatives”, CarbohydrateResearch (1997), 300(2), 139-142.

Preparation of 444

To a mixture of 443 (10 g, 47.39 mmol) and ketone 7 (13 g, 24 mmol) inanhydrous toluene (150 mL), is added PTSA (0.825 g, 0.2 eq) and heatedto reflux at 130° C. bath using dean stark apparatus for 24 hr. Cool thereaction mixture to room temperature and wash with sodium bicarbonatesolution. Crude product is purified by chromatography to get the product444.

Preparation of 445

To a solution of 444 in EtOH, Dimethylamine in EtOH is added and heatthe reaction mixture in a pressure reactor at 70° C. for overnight toget 445.

Example 51 Preparation of Compound 449

Preparation of 446

Compound 446 is prepared according to the procedure described in Persky,Rachel; Albeck, Amnon. “Synthesis of Selectively Labeled D-Fructose andD-Fructose Phosphate Analogues Locked in the Cyclic Furanose Form”.Journal of Organic Chemistry (2000), 65(18), 5632-5638.

Preparation of 449

Compound 447 is prepared using the procedure described above. It is thenconvert to the corresponding dimesylate 448 using standard method.Diamination of the dimesylate is carried out using dimethylamine in EtOHto get the compound 449.

Example 52 Synthesis of Zwitterionic Lipids

Example 53 Synthesis of Lipids Bearing Unsymmetric Lipid Anchors

Using a similar procedure used for the dilinoleyl ketone tail bearingcationic lipids, the unsymetric ketones 25, 27, 29, 31, 33 and 35 areall treated with the diols 517, 524, 528, 532, 536, 551, 554, 25 and 29to isolate the product with the unsymmetrical chains are isolated.

Example 54 Oigonucleotide Synthesis

All oligonucleotides were synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG, 500Å, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N-2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditewere purchased from (Promega). All phosphoramidites were used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichwas used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes was used. The activator was 5-ethyl thiotetrazole(0.75M, American International Chemicals), for the PO-oxidationIodine/Water/Pyridine was used and the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) was used.

3′-ligand conjugated strands were synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence was performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol was tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled siRNAs were synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite were purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block Anextended 15 min coupling of 0.1M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid bound oligonucleotide. Oxidation of the internucleotidephosphite to the phosphate was carried out using standard iodine-wateras reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate was introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent The cholesterol phosphoramidite was synthesized in house, andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite was 16 minutes.

After completion of synthesis, the support was transferred to a 100 mlglass bottle (VWR). The oligonucleotide was cleaved from the supportwith simultaneous deprotection of base and phosphate groups with 80 mLof a mixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at55° C. The bottle was cooled briefly on ice and then the ethanolicammonia mixture was filtered into a new 250 ml bottle. The CPG waswashed with 2×40 mL portions of ethanol/water (1:1 v/v). The volume ofthe mixture was then reduced to ˜30 ml by roto-vap. The mixture was thenfrozen on dyince and dried under vacuum on a speed vac.

The dried residue was resuspended in 26 ml of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionwas then quenched with 50 ml of 20 mM sodium acetate and pH adjusted to6.5, and stored in freezer until purification.

The oligoncuelotides were analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

The ligand conjugated oligonucleotides were purified reverse phasepreparative HPLC. The unconjugated oligonucleotides were purified byanion-exchange HPLC on a TSK gel column packed in house. The bufferswere 20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mMsodium phosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides were pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess werediluted in water to 150 μl and then pipetted in special vials for CGEand LC/MS analysis. Compounds were finally analyzed by LC-ESMS and CGE.

For the preparation of siRNA, equimolar amounts of sense and antisensestrand were heated in 1×PBS at 95° C. for 5 min and slowly cooled toroom temperature. Integrity of the duplex was confirmed by HPLC analysis

TABLE 7  siRNA duplexes for Luc and FVII targeting. SEQ ID  DuplexSense/Antisense Sequence 5′-3′ NO:  Target 1000/2434CUU ACG CUG AGU ACU UCG AdTdT Luc U*CG AAG fUAC UCA GCG fUAA GdT*dT2433/1001 C*UfU ACG CUG AGfU ACU UCG AdT*dT LucUCG AAG UAC UCA GCG UAA GdTdT 2433/2434C*UfU ACG CUG AGfU ACU UCG AdT*dT Luc U*CG AAG fUAC UCA GCG fUAA GdT*dT1000/1001 CUU ACG CUG AGU ACU UCG AdTdT LucUCG AAG UAC UCA GCG UAA GdTdT AD- GGAUCAUCUCAAGUCUUACdTdT FVII 1596GUAAGACUUGAGAUGAUCCdTdT AD- GGAfUfCAfUfCfUfCAAGfUfCfUfUAfCdTsdT FVII1661 GfUAAGAfCfUfUGAGAfUGAfUfCfCdT*dT Note: L8 is

lowercase is 2′-O-methyl modified nucleotide, * is phosphorothioatebackbone linkages, fN is a 2′-fluoro nucleotide, dN is 2′-deoxynucleotide.

Example 55 Serum Stability Assay for siRNA

A medium throughput assay for initial sequence-based stability selectionwas performed by the “stains all” approach. To perform the assay, ansiRNA duplex was incubated in 90% human serum at 37° C. Samples of thereaction mix were quenched at various time points (at 0 min., 15, 30,60, 120, and 240 min) and subjected to electrophoretic analysis (FIG.1). Cleavage of the RNA over the time course provided informationregarding the susceptibility of the siRNA duplex to serum nucleasedegradation.

A radiolabeled dsRNA and serum stability assay was used to furthercharacterize siRNA cleavage events. First, a siRNA duplex was 5′end-labeled with ³²P on either the sense or antisense strand. Thelabeled siRNA duplex was incubated with 90% human serum at 37° C., and asample of the solution was removed and quenched at increasing timepoints. The samples were analyzed by electrophoresis.

Example 56 FVII In Vivo Evaluation Using the Cationic Lipid DerivedLiposomes In vivo rodent Factor VII and ApoB Silencing Experiments

C57BL/6 mice (Charles River Labs, MA) and Sprague-Dawley rats (CharlesRiver Labs, MA) received either saline or siRNA in desired formulationsvia tail vein injection at a volume of 0.01 mL/g. At various time pointspost-administration, animals were anesthesized by isofluorane inhalationand blood was collected into serum separator tubes by retro orbitalbleed. Serum levels of Factor VII protein were determined in samplesusing a chromogenic assay (Coaset Factor VII, DiaPharma Group, OH orBiophen FVII, Aniara Corporation, OH) according to manufacturerprotocols. A standard curve was generated using serum collected fromsaline treated animals. In experiments where liver mRNA levels wereassessed, at various time points post-administration, animals weresacrificed and livers were harvested and snap frozen in liquid nitrogen.Frozen liver tissue was ground into powder. Tissue lysates were preparedand liver mRNA levels of Factor VII and apoB were determined using abranched DNA assay (QuantiGene Assay, Panomics, Calif.).

Example 57 In Vivo Evaluation of FVII siRNA 1661 Using Cyclic CationicLipids, 506, 512 and 519 Based Formulation

The cyclic cationic lipids 506, 512 and 519-based formulations asdescribed below were prepared, characterized and evaluated in vivo byFVII assay. Results show that lipid particles formed with 519 showedalmost complete knockdown of FVII protein levels in vivo.

519/Chol/PEG-C14/DSPC 506/Chol/PEG-C14/DSPC 512/Chol/PEG-C14/DSPC52:30:5:13 52:30:5:13 52:30:5:13 Particle size before siRNA 87.4 96.5102 conjugation(nm) PDI 0.155 0.164 0.183 Particle size final (nm) 81 8895.8 PDI 0.105 0.1206 0.119 % Encapsulation, 91.5 64 87.5 Ribogreen

Example 58 Liposome Formulations for FVII Targeting

The formulations shown above are made with DPPC in place of DSPC withvarying amounts and the assays are repeated.

519/Chol/PEG-C14/DPPC 512/Chol/PEG-C14/DPPC; 506/Chol/PEG-14/DPPC;52:30:5:13 52:30:5:13 52:30:5:13

Example 59 Preparation of 1,2-Di-O-alkyl-sn3-Carbomoylglyceride(PEG-DMG)

Preparation of IVa

1,2-Di-O-tetradecyl-sn-glyceride Ia (30 g, 61.80 mmol) andN,N′-succinimidylcarboante (DSC, 23.76 g, 1.5 eq) were taken indichloromethane (DCM, 500 mL) and stirred over an ice water mixture.Triethylamine (TEA, 25.30 mL, 3 eq) was added to the stirring solutionand subsequently the reaction mixture was allowed to stir overnight atambient temperature. Progress of the reaction was monitored by TLC. Thereaction mixture was diluted with DCM (400 mL) and the organic layer waswashed with water (2×500 mL), aqueous NaHCO₃ solution (500 mL) followedby standard workup. The residue obtained was dried at ambienttemperature under high vacuum overnight. After drying the crudecarbonate IIa thus obtained was dissolved in dichloromethane (500 mL)and stirred over an ice bath. To the stirring solution mPEG₂₀₀₀-NH₂(III, 103.00 g, 47.20 mmol, purchased from NOF Corporation, Japan) andanhydrous pyridine (Py, 80 mL, excess) were added under argon. Thereaction mixture was then allowed to stir at ambient temperatureovernight. Solvents and volatiles were removed under vacuum and theresidue was dissolved in DCM (200 mL) and charged on a column of silicagel packed in ethyl acetate. The column was initially eluted with ethylacetate and subsequently with gradient of 5-10% methanol indichloromethane to afford the desired PEG-Lipid IVa as a white solid(105.30 g, 83%). ¹H NMR (CDCl₃, 400 MHz)=5.20-5.12 (m, 1H), 4.18-4.01(m, 2H), 3.80-3.70 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂),2.10-2.01 (m, 2H), 1.70-1.60 (m, 2H), 1.56-1.45 (m, 4H), 1.31-1.15 (m,48H), 0.84 (t, J=6.5 Hz, 6H). MS range found: 2660-2836.

Preparation of IVb

1,2-Di-O-hexadecyl-sn-glyceride Ib (1.00 g, 1.848 mmol) and DSC (0.710g, 1.5 eq) were taken together in dichloromethane (20 mL) and cooleddown to 0° C. in an ice water mixture. Triethylamine (1.00 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solutionand dried over sodium sulfate. Solvents were removed under reducedpressure and the resulting residue of IIb was maintained under highvacuum overnight. This compound was directly used for the next reactionwithout further purification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol,purchased from NOF Corporation, Japan) and IIb (0.702 g, 1.5 eq) weredissolved in dichloromethane (20 mL) under argon. The reaction wascooled to 0° C. Pyridine (1 mL, excess) was added and the reactionstirred overnight. The reaction was monitored by TLC. Solvents andvolatiles were removed under vacuum and the residue was purified bychromatography (first ethyl acetate followed by 5-10% MeOH/DCM as agradient elution) to obtain the required compound IVb as a white solid(1.46 g, 76%). ¹H NMR (CDCl₃, 400 MHz) δ=5.17 (t, J=5.5 Hz, 1H), 4.13(dd, J=4.00 Hz, 11.00 Hz, 1H), 4.05 (dd, J=5.00 Hz, 11.00 Hz, 1H),3.82-3.75 (m, 2H), 3.70-3.20 (m, —O—CH₂—CH₂—O—, PEG-CH₂), 2.05-1.90 (m,2H), 1.80-1.70 (m, 2H), 1.61-1.45 (m, 6H), 1.35-1.17 (m, 56H), 0.85 (t,J=6.5 Hz, 6H). MS range found: 2716-2892.

Preparation of IVc

1,2-Di-O-octadecyl-sn-glyceride Ic (4.00 g, 6.70 mmol) and DSC (2.58 g,1.5 eq) were taken together in dichloromethane (60 mL) and cooled downto 0° C. in an ice water mixture. Triethylamine (2.75 mL, 3 eq) wasadded and the reaction was stirred overnight. The reaction was followedby TLC, diluted with DCM, washed with water (2 times), NaHCO₃ solution,and dried over sodium sulfate. Solvents were removed under reducedpressure and the residue was maintained under high vacuum overnight.This compound was directly used for the next reaction without furtherpurification. MPEG₂₀₀₀-NH₂ III (1.50 g, 0.687 mmol, purchased from NOFCorporation, Japan) and IIc (0.760 g, 1.5 eq) were dissolved indichloromethane (20 mL) under argon. The reaction was cooled to 0° C.Pyridine (1 mL, excess) was added and the reaction was stirredovernight. The reaction was monitored by TLC. Solvents and volatileswere removed under vacuum and the residue was purified by chromatography(ethyl acetate followed by 5-10% MeOH/DCM as a gradient elution) toobtain the desired compound IVc as a white solid (0.92 g, 48%). ¹H NMR(CDCl₃, 400 MHz) δ=5.22-5.15 (m, 1H), 4.16 (dd, J=4.00 Hz, 11.00 Hz,1H), 4.06 (dd, J=5.00 Hz, 11.00 Hz, 1H), 3.81-3.75 (m, 2H), 3.70-3.20(m, —O—CH₂—CH₂—O—, PEG-CH₂), 1.80-1.70 (m, 2H), 1.60-1.48 (m, 4H),1.31-1.15 (m, 64H), 0.85 (t, J=6.5 Hz, 6H). MS range found: 2774-2948.

Example 60 Preparation of Compound 563 (ALNY-106)

Preparation of Compound 557

An ice cold solution of but-3-en-1-amine hydrochloride (5 g, 46.5 mmol)and NEt₃ (2.4 eq) in DCM (300 mL) was treated dropwise with compound2-Nos-Cl (1.1 eq) in DCM (120 mL) then warmed to r.t. After 2 h, aqueousworkup then column chromatography gave compound 557 (11.6 g, 97%). ¹HNMR (CDCl₃, 400 MHz) δ=8.22-8.06 (m, 1H), 7.87 (dt, J=6.5, 3.0, 1H),7.81-7.64 (m, 2H), 5.65 (ddt, J=17.1, 10.3, 6.9, 1H), 5.32 (d, J=17.6,1H), 5.13-4.96 (m, 2H), 3.19 (q, J=6.5, 2H), 2.28 (q, J=6.7, 2H);Electrospray MS (+ve): Molecular weight for C10H12N2O4S (M+H)⁺ Calc.257.1; Found 257.0.

Preparation of Compound 558

An ice-cold solution of but-3-en-1-ol (3.6 g, 50 mmol) and PPh₃ (1.2 eq)in DCM (400 mL) was treated dropwise over 15 min with DIAD (1.1 eq).After a further 5 min, a solution of compound 557 (11.6 g, mmol) in DCM(60 mL) was added dropwise over 10 min and the resulting solution wasallowed to warm to r.t. The solvent was removed. Column chromatographygave pure compound 558 (13.1 g, 93%). ¹H NMR (400 MHz, CDCl₃) δ8.11-7.93 (m, 1H), 7.68 (pd, J=7.4, 3.8, 2H), 7.64-7.58 (m, 1H), 5.69(ddt, J=17.1, 10.2, 6.8, 2H), 5.06 (dd, J=17.2, 1.5, 2H), 5.02 (d,J=10.2, 2H), 3.48-3.30 (m, 4H), 2.30 (dd, J=14.9, 7.1, 4H); ElectrosprayMS (+ve): Molecular weight for C14H18N2O4S (M+H)⁺ Calc. 311.1; Found311.0.

Preparation of Compound 559

A heated (50° C.) solution of 558 (3 g, 9.66 mmol) in ACN (100 mL) wastreated successively with thioglycolic acid (3 eq) and NaOMe (25 w/w %solution in MeOH, 6 eq). Heating was continued for 1 h, then cooled tor.t. Aqueous workup gave the crude deprotected secondary amine whichused without purification and subjected to Cbz protection by reactionwith Cbz-OSu (1.5 eq) in the presence of NEt₃ (2 eq) at r.t. overnight.Aqueous workup then column chromatography gave compound 559 (1.65 g,66%). ¹H NMR (400 MHz, CDCl₃) δ ¹H NMR (400 MHz, DMSO) δ 7.44-7.23 (m,5H), 5.85-5.62 (m, 2H), 5.09-4.90 (m, 6H), 3.26 (t, J=7.2, 4H), 2.23 (q,J=6.6, 4H); Electrospray MS (+ve): Molecular weight for C16H21NO2 (M+H)⁺Calc. 260.2; Found 260.0.

Preparation of Compound 560

A DCM solution of compound 559 (1.65 g, 6.36 mmol) was treated withGrubbs second generation catalyst (1 mol %) at r.t. for 18 h. Columnchromatography gave compound 560 (1.29 g, 88%). ¹H NMR (400 MHz, CDCl₃)δ 7.44-7.13 (m, 5H), 5.73 (q, J=3.6, 2H), 5.15 (s, 2H), 3.63-3.43 (m,4H), 2.31 (dd, J=19.2, 4.2, 4H); Electrospray MS (+ve): Molecular weightfor C14H17NO2 (M+H)⁺ Calc. 232.1; Found 232.0.

Preparation of Compound 561

A solution of compound 560 (1.29 g, 5.58 mmol) in acetone (10 mL) wasadded in a dropwise fashion to a stirring aqueous solution (10 mL) ofNMO (1.5 eq) and OsO₄ (1 mol %). After 18 h at r.t., TLC indicated acomplete reaction. The solvent was removed. Aqueous workup then columnchromatography gave pure compound 561 (1.28 g, 86%). ¹H NMR (400 MHz,DMSO) δ 7.48-7.25 (m, 5H), 5.09 (s, 2H), 4.56 (d, J=4.2, 2H), 3.67 (d,J=3.5, 2H), 3.45 (ddd, J=14.3, 7.4, 3.7, 2H), 3.40-3.18 (m, 2H), 1.86(dtd, J=11.5, 7.4, 3.7, 2H), 1.70-1.49 (m, 2H); Electrospray MS (+ve):Molecular weight for C14H19NO4 (M+H)⁺ Calc. 266.1; Found 266.0.

Preparation of Compound 562

A solution of dilinoleyl ketone (1.05 eq), PTSA (0.1 eq) and compound561 (1.28 g, 4.82 mmol) in toluene (200 mL) was brought to reflux for 2h. Removal of solvent then column chromatography gave pure compound 562(1.44 g, 39%). ¹H NMR (400 MHz, CDCl₃) δ 7.49-7.14 (m, 5H), 5.52-5.23(m, 8H), 5.13 (s, 2H), 4.39 (s, 2H), 3.75-3.53 (m, 2H), 3.44 (dd,J=14.3, 9.3, 2H), 2.77 (t, J=6.4, 4H), 2.05 (dd, J=13.6, 6.7, 10H), 1.80(s, 2H), 1.69 (dd, J=10.2, 6.1, 2H), 1.66-1.51 (m, 2H), 1.51-1.09 (m,36H), 0.89 (t, J=6.8, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 155.64, 136.89,130.17, 130.16, 130.13, 130.09, 128.45, 127.96, 127.92, 127.89, 127.78,110.14, 76.14, 67.08, 42.41, 41.99, 36.54, 34.95, 32.93, 32.48, 31.51,29.97, 29.93, 29.65, 29.54, 29.46, 29.34, 29.29, 27.22, 27.21, 27.18,25.61, 24.53, 23.64, 22.56, 14.07; Electrospray MS (+ve): Molecularweight for C51H83NO4 (M+Na)⁺ Calc. 796.6; Found 796.4.

Preparation of Compound 563

A solution of compound 562 (1.4 g, 1.8 mmol) in hexane (20 mL) was addedin a dropwise fashion over 10 min to an ice-cold solution of LAH in THF(1 M, 3.6 mL, 2 eq). On complete addition, the mixture was heated at 40°C. over 0.5 h then ice-cooled again. Aqueous workup then columnchromatography gave compound 4 (1.04 g, 88%) as a colorless oil. ¹H NMR(400 MHz, CDCl₃) δ 5.48-5.23 (m, 8H), 4.43-4.24 (m, 2H), 2.85-2.64 (m,6H), 2.30 (s, 3H), 2.27-2.17 (m, 2H), 2.04 (q, J=6.6, 8H), 2.00-1.84 (m,4H), 1.65 (dd, J=10.1, 6.0, 2H), 1.60-1.49 (m, 2H), 1.47-1.16 (m, 36H),0.88 (t, J=6.8, 6H); ¹³C NMR (101 MHz, CDCl₃) δ 130.39, 130.34, 128.16,128.12, 111.09, 53.90, 47.34, 36.84, 36.30, 31.74, 31.25, 30.23, 30.12,29.90, 29.88, 29.86, 29.79, 29.74, 29.70, 29.57, 29.55, 29.52, 27.47,27.44, 27.41, 25.84, 24.72, 23.63, 22.79, 14.30; Electrospray MS (+ve):Molecular weight for C44H79NO2 (M+Na)⁺ Calc. 654.6; Found 654.4.

Example 61 Preparation of Compounds 571 and 573 (ALNY-121 and ALNY-122)

Preparation of Compound 567

An ice-cold solution of compound 564 (5.56 g, 49.6 mmol) and NEt₃ (1.2eq) in DCM (300 mL) was treated in a dropwise fashion over 5 min withMsCl (1.1 eq). After 1 h at 0° C., aqueous workup gave crude compound565 (pure by TLC) which was used without purification in the next step.

Compound 565 (49.6 mmol) was treated with 33% H₂NMe in EtOH (100 mL)over 72 h. Excess H₂NMe was removed by co-evaporation with Et₂O, thenthe ethanolic solution of compound 566 was treated with conc. HCl (2eq). The EtOH was removed by evaporation and the residue was redissolvedin DCM (100 mL). NEt₃ (6 eq) was added followed by Cbz-OSu (2 eq) andthe mixture was stirred at r.t. for 3 h. Aqueous workup then columnchromatography gave pure compound 568 as an oil (11.3 g, 88% from 564).¹H NMR (400 MHz, CDCl₃) δ 7.49-6.97 (m, 5H), 5.66 (s, 2H), 5.13 (s, 2H),3.21 (t, J=8.1, 2H), 2.94 (d, J=2.8, 3H), 2.30-1.82 (m, 4H), 1.82-1.53(m, 2H), 1.37-0.97 (m, 1H).

Preparation of Compound 568 and 569

Using a procedure which is analogous to those described for thesynthesis of compounds 504, a mixture of 568 and 569 was obtained. Thismixture was then separated by preparative HPLC to give both pureisomers.

Preparation of Compound 570 and 572

Using a procedure analogous to that described for the synthesis ofcompound 505, compounds 570 and 572 are obtained as colorless oils.

Preparation of Compound 571 and 573

Using a procedure analogous to that described for the synthesis ofcompound 506, compounds 571 and 573 are obtained as colorless oils.

Example 62 Preparation of Compound 581 and 583 (ALNY-115 & ALNY-116)

Preparation of Compound 577

An ice-cold solution of compound 574 (1.05 g, 10.7 mmol) and NEt₃ (1.2eq) in DCM (50 mL) was treated in a dropwise fashion over 5 min withMsCl (1.1 eq). After 1 h at 0° C., aqueous workup gave crude compound575 (1.88 g, 99%, pure by TLC) which was used without purification inthe next step.

Compound 575 (10.5 mmol) was treated with 33% H₂NMe in EtOH (100 mL) at50° C. over 18 h. Excess H₂NMe was removed by co-evaporation with Et₂O,then the ethanolic solution of compound 576 was treated with conc. HCl(2 eq). The EtOH was removed by evaporation and the residue wasredissolved in DCM (100 mL). NEt₃ (4 eq) was added followed by Cbz-OSu(1.5 eq) and the mixture was stirred at r.t. for 3 h. Aqueous workupthen column chromatography gave pure compound 577 as an oil (86% from574). ¹H NMR (400 MHz, CDCl₃) δ 7.54-7.04 (m, 5H), 5.65 (d, J=8.6, 2H),5.13 (s, 2H), 3.27 (t, J=8.9, 2H), 2.94 (s, 3H), 2.60 (s, 1H), 2.51-2.28(m, 2H), 2.04 (dd, J=22.1, 15.3, 2H); ¹³C NMR (101 MHz, CDCl₃) δ 129.47,128.40, 127.83, 127.75, 66.92, 53.74, 53.13, 38.92, 36.34, 36.23, 36.06,35.78; Electrospray MS (+ve): Molecular weight for C15H19NO2 (M+H)⁺Calc. 246.1; Found 246.0.

Preparation of Compound 578 and 579

Using a procedure which is analogous to those described for thesynthesis of compounds 504, a mixture of 578 and 579 was obtained. Thismixture was then separated by preparative HPLC to give both pureisomers.

Preparation of Compound 580 and 582

Using a procedure analogous to that described for the synthesis ofcompound 505, compounds 580 and 582 are obtained as colorless oils.

Preparation of Compound 581 and 583

Using a procedure analogous to that described for the synthesis ofcompound 506, compounds 581 and 583 are obtained as colorless oils.

Example 63

Example 64

Example 65

Synthesis of 2001: To 500 ml of ethanol cooled below 0° C. usingice-salt mixture was added 10 ml of Conc. H₂SO₄ slowly. Linoleic acid(100 g, 357 mmol) in 500 ml of ethanol was added to the above solutionslowly by maintaining the temperature below 0° C. After addition thereaction mass was warmed to RT and then refluxed for 5 hrs (TLC). It wasthen cooled to room temperature and neutralized by sat. NaHCO₃ solution.The resulting solution was concentrated to remove excess of solvent. Theresidue was diluted with water (1000 ml) and extracted with DCM (6×500ml). The combined organic layer was washed with brine (1000 ml), anddried over sodium sulfate, filtered and evaporated at reduced pressureto obtain pure product (109.80 g, 99%) as a pale yellow liquid, whichwas taken as such for the next stage. ¹H NMR (CDCl₃): δ 0.89 (t, 3H,J=6.8 Hz), 1.24-1.31 (m, 17H), 1.62 (m, 2H, J=10 Hz), 2.04 (q, 4H,J₁=6.8 Hz, J₂=6.8 Hz), 2.29 (t, 2H, J=7.6 Hz), 2.76 (t, 2H, J=6.4 Hz),4.13 (q, 2H, J₁=7.2 Hz, J₂=7.2 Hz), 5.34 (m, 4H). ¹³C NMR (CDCl₃): δ13.9, 14.1, 22.5, 24.9, 25.5, 27.1, 29.0, 29.1, 29.3, 29.5, 31.4, 34.2,60.0, 127.8, 127.9, 129.9, 130.0, 173.6.

Synthesis of 2002: To 660 ml of freshly distilled toluene in a 2 Lmultineck RB flask fitted with reflux condenser under argon was addedsodium pieces (41.1 g, 1.785 mol). To this was added TMSCl (192 mL,1.499 mol) slowly and heated to 40° C. after addition. Then a solutionof 2001 (110 g, 0.357 mol) in 275 ml of freshly distilled toluene wasadded slowly by maintaining the reaction temperature at 40° C. over aperiod of 1 hr. It was then refluxed for 2-3 hrs. After 3 hrs thereaction mass turned pale purple in color (TLC). Heating was stopped andthe reaction mass was cooled to room temperature, filtered through a padof celite and washed with toluene. (CAUTION: The reaction mixturecontained unreacted sodium pieces). The filtrate obtained was stirredwith 3 L of sat. NH₄Cl solution for 15-20 minutes until the silyl etherconverted to the required α-keto alcohol. The organic layer wasseparated and the aqueous layer was washed with ethyl acetate (3×1000ml). The combined organic layer was washed with brine (1 L), dried oversodium sulfate, filtered and evaporated at reduced pressure to obtainthe crude material, which was purified by silica gel chromatographyusing hexane/ethyl acetate as eluent. The product got eluted at 3% ethylacetate in hexane to get 2002 (44 g, 47%) as a pale yellow liquid. ¹HNMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J=7.2 Hz), 1.2-1.3 (m, 30H), 1.53(m, 1H), 1.62 (m, 2H), 1.8 (m, 1H), 2.04 (q, 8H, J₁=6.8 Hz, J₂=6.8 Hz),2.43 (m, 2H), 2.76 (t, 4H, J=6.4 Hz), 3.49 (d, 1H, J=4.8 Hz) 4.16 (m,1H), 5.34 (m, 8H). ¹³C NMR (100 MHz, CDCl₃): δ 14.1, 22.6, 23.6, 24.8,25.6, 27.2, 29.1, 29.2, 29.24, 29.3, 29.4, 29.56, 29.6, 31.5, 33.7,37.8, 76.3, 127.8, 127.9, 129.9, 130.0, 212.4.

Synthesis of 2003: A solution of 2002 (44 g, 83 mol) in methanol/DCMmixture (490 mL, DCM was added to make the solution homogeneous) underargon was cooled below 0° C. using ice-salt mixture. Sodium borohydride(4.7 g, 125 mmol) was added in one lot to the reaction mass. Thesuspension was stirred for 2 hrs, and the mass temperature slowly raisedto RT. After 2 hrs the reaction mass became homogeneous. TLC showed theabsence of starting material. The reaction was quenched with 100 ml ofwater, and concentrated to remove excess solvent. The residue obtainedwas again diluted with water (500 ml), and extracted with DCM (4×500ml). The combined organic layer was washed with brine (500 ml), driedover sodium sulfate, filtered and evaporated at reduced pressure toobtain the crude product, which was purified by silica gelchromatography using hexane/ethyl acetate as eluent. The product elutedfrom 4% to 20% of ethyl acetate in hexane to get 2003 (36 g, 82%) as awhite semisolid. ¹H NMR (CDCl₃): δ 0.89 (t, 6H, J=6.8 Hz), 1.2-1.5 (m,36H), 1.78 (d, 1H, J=4 Hz), 1.95 (d, 1H, J=4 Hz), 2.04 (q, 8H, J₁=6.8Hz, J₂=6.8 Hz), 2.77 (t, 4H, J=6.4 Hz), 3.40 (m, 1H), 3.61 (m, 1H), 5.34(m, 8H). ¹³C NMR (CDCl₃): δ13.7, 22.2, 25.2, 25.3, 25.7, 26.8, 28.4,28.9, 29.0, 29.1, 29.3, 30.8, 31.1, 33.2, 74.1, 74.3, 127.5, 127.6,129.7, 129.8. MS: Molecular weight calculated for C₃₆H₆₆O₂ 530.51;Found: 531.52 (M+H)

Example 66

Synthesis of 2005: To a solution of 2004 (50 g, 95 mmol) in DCM (400 ml)under Ar atmosphere, was added TEA (53 mL, 378 mmol) and DMAP (1.2 g,9.5 mmol) and stirred at room temperature under Ar atmosphere. Reactionmass was cooled to −5° C. and the solution of mesyl chloride (15 mL, 190mmol) in DCM (100 ml) was added slowly at temperature below −5° C. andallowed to warm to RT after addition. After 30 minutes (TLC), reactionmass was quenched with ice cold water (20 ml). Organic layer wasseparated, washed with 1N HCl (30 ml), water, brine, dried over sodiumsulfate and evaporated at reduced pressure to obtain pure product (55 g,95.5%) as yellow liquid. 1H NMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J=6.8),1.2-1.5 (m, 36H), 1.67 (m, 4H), 2.05 (q, 8H, J1=6.8, J2=6.8), 2.77 (t,4H, J=6.4), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m, 8H).

Synthesis 2006: To a solution of 2005 (50 g, 82 mmol) in DMF (500 mL)under argon atmosphere, was added NaN₃ (27 g, 410 mmol) and heated to70° C. and maintained the temperature for four hours (TLC). The mixturewas diluted with water and extracted with ethyl acetate (3×250 ml). Theorganic layer was washed with water, brine, dried over Na₂SO₄ andevaporated at reduced pressure to give crude product, which was purifiedby silica gel chromatography using hexane/ether as eluent. The productwas eluted at 2% ether hexane to get 2006 (36 g, 86%) as pale yellowliquid. ¹H NMR (400 MHz, CDCl3): δ 0.90 (t, 8H), 1.30 (m, 36H), 1.49 (t,4H, J=6.4 Hz) 2.04 (q, 8H, J1=7.6, J2=14 Hz), 2.77 (t, 4H, J=6.4 Hz),3.22 (m, 1H), 5.34 (m, 8H). ¹³C NMR (400 MHz, CDCl₃): δ 14.1, 22.5,25.6, 26.1, 27.2, 29.2, 29.3, 29.45, 29.65, 31.5, 34.1, 63.1, 127.9, and130.1. IR (KBr): 2098.

Example 67

Synthesis of 2007: To a solution of 2005 (76 g, 125 mmol) indimethylformamide (500 mL), was added sodium hydrosulfide hydrate (35 g,625 mmol) at room temperature. Reaction mixture was heated to 70° C. for2 hrs (TLC). It was then cooled to room temperature and diluted withwater (7V) and extracted with ether (3×5V). Combined ether layer waswashed with water (2×3V), brine solution (2×3V), dried over sodiumsulfate and evaporated at reduced pressure to obtain the crude product,which was purified by silica gel chromatography using a hexane as eluentto get the product 2007 (43.6 g, 64%). MS: Molecular weight calculatedfor C₃₇H₆₈S 544.50; Found: 545.51 (M+H).

Synthesis of 2008: To a solution of aldrithiol (20.2 g, 92 mmol) indichloromethane (400 ml) was added benzyl bromide (11 mL, 92 mmol) at 0°C. After stirring at 0° C. for 15 min, it was warmed to room temperatureand stirred for 15 minutes. Reaction mixture was cooled back to 0° C.and added a solution of 2007 (50 g, 92 mmol) in dichloromethane (100 ml)followed by diisopropylethylamine (16 mL, 92 mmol). After addition, itwas heated to reflux for 2 hrs (TLC). It was then diluted withdichloromethane (10V), washed with water (2×10V), brine solution(2×10V), dried over sodium sulfate and evaporated at reduced pressure toobtain crude product, which was purified by silica gel chromatographyusing 3% ether/hexane to afford pure product as pale yellow liquid. (35g, 58%) ¹H NMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J1=6.4 Hz, J2=7.2 Hz),1.25-1.42 (m, 38H), 1.56-1.63 (m, 2H), 2.05 (q, 8H, J1=6.4 Hz, J2=14Hz), 2.78 (t, 5H, J1=6.4 Hz, J2=6 Hz), 5.30-5.42 (m, 8H), 7.06 (t, 1H,J1=5.2 Hz, J2=6.8 Hz), 7.62 (t, 1H, J1=7.6 Hz, J2=7.6 Hz), 7.76 (d, 1H,J=8 Hz), 8.42 (d, 1H, J=4.4 Hz). ¹³C NMR (100 MHz, CDCl₃): δ 22.6, 25.6,26.7, 27.2, 29.2, 29.3, 29.5, 29.6, 31.5, 33.74, 52.9, 119.9, 120.4,127.9, 128, 130.1, 130.2, 136.7, 149.3, 161.5. MS: Molecular weightcalculated for C₄₂H₇₁NS₂ 653.50; Found: 654.49 (M+H).

Example 68

Synthesis of 2009 (ALNY-138): A solution of 2005 (5 g, 8 mmol) in DMFand dimethylamine—40% aqueous solution was taken in a seal tube. Thereaction mixture was heated at 90° C. for 20 hours (TLC). It was thencooled to room temperature, poured to water and extracted with ethylacetate (3×50 ml). The organic layer was washed with water & brine,dried over Na₂SO₄ and evaporated to afford pure product as pale brownliquid (2.00 g, 45%) ¹H NMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, j=6.8),1.2-1.4 (m, 40H), 2.05 (q, 8H, J₁=6.8 Hz, J₂=6.8 Hz), 2.2 (s, 6H), 2.77(t, 4H, j=6.4 Hz), 5.35 (m, 8H). ¹³C NMR (400 MHz, CDCl₃): δ 14.1, 22.5,22.6, 27.1, 27.2, 29.3, 29.5, 29.57, 29.63, 29.67, 30.0, 31.5, 32.5,40.5, 64.0, 127.9 and 130.1 MS: Molecular weight calculated for C₃₉H₇₃N555.57; Found: 556.55 (M+H).

Example 69

Synthesis of 2010: To a solution of 2004 (30 g, 56.8 mmol) in toluene,was added N-Hydroxyphthalimide (13.9 g, 85 mmol) and TPP (22.30 g, 85mmol) under argon. The reaction mass was cooled to −5° C., to this wasadded TEA (11.84 mL), followed by DEAD (13.14 ml). The reaction mass wasallowed to stir for 12 hrs at room temperature (TLC). It was thenfiltered through celite pad. The filtrate was evaporated at reducedpressure to obtain crude product, which was purified by silica gelchromatography to afford pure product, which was eluted at 3% diethylether and hexane to get the product 2010 (22.90 g, 60.50%) as paleyellow liquid ¹HNMR (400 MHz, CDCl₃,): δ 0.90 (6H, t, J=7.2 Hz), 1.2-1.4(34H, m), 1.66-1.70 (4H, m), 2.03-2.08 (8H, m), 2.78 (4H, t, J=12.8 Hz),4.22 (1H, m), 5.29-5.43 (8H, m), 7.74-7.76 (2H, m), 7.83-7.85 (2H, m).¹³CNMR (100 MHz, CDCl₃,): δ 14.3, 22.5, 24.9, 25.6, 27.2, 27.20, 29.3,29.3, 29.5, 29.5, 29.6, 29.7, 31.5, 32.4, 88.3, 123.3, 127.9, 129.0,130.1, 134.3, 164.3. MS: Molecular weight calculated for C₄₅H₇₁NO₃673.54; Found: 674.55 (M+H).

Example 70

Example 71

Example 72 Synthesis of 2019 (ALNY-152)

Example 73 Synthesis of 2022 (ALNY-153)

Example 74 Synthesis of 2025 (ALNY-158)

Example 75 Synthesis of 2028 (ALNY-156)

Example 76 Synthesis of 2031 (ALNY-157)

Example 77 Synthesis of 2035

Example 78 Synthesis of3-(dimethylamino)-N-((11Z,14Z)-2-((9Z,12Z)-octadeca-9,12-dienyl)icosa-11,14-dienyl)propanamide(ALNY-201)

To a stirred suspension of N,N-dimethylamino propionic acidhydrochloride (1, 0.198 g, 1.3 mmol, 1.0 eq) in DCM was added HBTU (0.59g, 1.56 mmol, 1.2 eq) and DIPEA (0.71 mL, 3.9 mmol, 3.0 eq) at roomtemperature. After stirred for 10 minutes, a solution of amine (2, 0.7g. 1.3 mmol, 1.0 eq) in DCM was added drop wise at room temperature andcontinued the stirring until completion of the reaction. Reactionmixture was diluted with DCM, washed with saturated NaHCO₃ solutionfollowed by brine, organic layer was separated and dried over MgSO₄,concentrated and purified by the silica gel column chromatography usingDCM:MeOH (5%) as gradients to get pure oily compound 3 in 70% yield. ¹HNMR (400 MHz, CDCl₃) δ 7.18 (brs, 1H), 5.47-5.19 (m, 8H), 3.18-3.07 (m,4H), 2.76 (t, J=6.5, 4H), 2.70 (s, 6H), 2.60 (t, J=6.0, 2H), 2.04 (q,J=6.8, 9H), 1.48 (brs, 1H), 1.40-1.14 (m, 43H), 0.88 (t, J=6.8, 6H).Calc. mass for the C43H80N2O: 640.6; found 641.5.

Synthesis of Novel Dilinoleyl Derivatives

No compound Name 1

ALNY-192 2

ALNY-200 3

ALNY-175 4

ALNY-187 5

ALNY-149 6

ALNY-202 Compound 1

Compound 2

Compound 3

Compound 4

Compound 5

Compound 6

Experimental Details Compound 1 (ALNY-192)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 3-dimethylamino-1-propanol (2.43 g, 23.6 mmol) wasadded dropwise. The resulting mixture was stirred at room temperatureovernight. Taken up 50 mL of the solution, Et₃N (0.822 mL, 5.90 mmol)and ALN-SAN-30 (2.08 g, 3.93 mmol) were added and the reaction mixturewas stirred at room temperature overnight. The reaction mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. The organiclayer was dried over anhydrous MgSO₄, filtered and concentrated. Thecrude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 1 (1.66 g, 2.53 mmol, 64%, R_(f)=0.22 with 5%MeOH in CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz) δ 5.30-5.41 (m, 8H), 4.37 (d,J=8.0 Hz, 1H), 4.09 (t, J=6.0 Hz, 2H), 3.57 (brs, 1H), 2.78 (t, J=6.0Hz, 4H), 2.33 (t, J=8.0 Hz, 2H), 2.23 (s, 6H), 2.02-2.06 (m, 8H),1.76-1.80 (m, 2H), 1.27-1.45 (m, 40H), 0.89 (t, J=8.0 Hz, 6H). ¹³C NMR(CDCl₃, 100 MHz) δ 156.5, 130.4, 130.3, 128.2, 128.1, 63.2, 56.6, 51.4,45.7, 35.7, 31.7, 29.9, 29.8, 29.7, 29.6, 29.5, 27.7, 27.5, 27.4, 26.0,25.8, 22.8, 14.3. Molecular weight for C₄₃H₈₁N₂O₂ (M+H)⁺ Calc. 657.63;Found 657.5.

Compound 2 (ALNY-200)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 3-dimethylamino-1-propanol (2.43 g, 23.6 mmol) wasadded dropwise. The resulting mixture was stirred at room temperatureovernight. Taken up 50 mL of the solution, Et₃N (0.697 mL, 5.00 mmol)and ALN-SAN-033 (1.71 g, 3.15 mmol) were added and the reaction mixturewas stirred at room temperature overnight. The reaction mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. The organiclayer was dried over anhydrous MgSO₄, filtered and concentrated. Thecrude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 2 (1.14 g, 1.70 mmol, 54%, R_(f)=0.13 with 5%MeOH in CH₂Cl₂). Molecular weight for C₄₄H₈₃N₂O₂ (M+H)⁺ Calc. 671.65;Found 671.5.

Compound 3 (ALNY-175)

To a flask containing EtOH (50 mL) was added dimethylaminoethylhydrazine dihydrochloride (1.00 g, 5.70 mmol) and ALNY-SAN-003 (2.00 g,3.80 mmol). The mixture was heated at 60° C. for 16 hours. Afteraddition of Et₃N (0.5 mL), the reaction mixture was evaporated. Theresidue was extracted with Et₂O and saturated NaHCO₃ aq., and theorganic layer was dried over MgSO₄, filtered and concentrated. The crudewas purified by silica gel column chromatography (CH₂Cl₂:MeOH:NH₃aq.=95:5:0.5, R_(f)=0.29) to give compound 3 (1.78 g, 2.91 mmol, 76%).Molecular weight for C₄₁H₇₈N₃ (M+H)⁺ Calc. 612.62; Found 612.5.

Compound 4 (ALNY-187)

3-Dimethylamino-propionic acid hydrazide (Ryan Scientific, 500 mg, 3.89mmol) in EtOH (10 mL) and the dilinoleyl ketone (1.74 g, 3.31 mmol) inEtOH (20 mL) were mixed together. To the solution was added acetic acid(0.038 mL, 0.662 mmol), and the reaction mixture was heated at 65° C.for 5 hours. After addition of Et₃N (0.5 mL), the reaction mixture wasevaporated. The residue was extracted with CH₂Cl₂ and saturated NaHCO₃aq., and the organic layer was dried over MgSO₄, filtered andconcentrated. The crude was purified by silica gel column chromatography(CH₂Cl₂:MeOH:NH₃ aq.=95:5:0.5, R_(f)=0.30) to give compound 4 (1.40 g,2.19 mmol, 66%). Molecular weight for C₄₂H₇₈N₃O (M+H)⁺ Calc. 640.61;Found 640.5.

Compound 5 (ALNY-149)

ALY-SAN-031 (2.36 g, 3.50 mmol) was treated with hydrazine monohydrate(0.424 mL, 5.60 mmol) in CH₂Cl₂ (36 mL) and EtOH (4 mL) for 2 hours.After filtration of the resulting white precipitation, the filtrate wasconcentrated. The residue was extracted with Et₂O and saturated NaHCO₃aq., and the organic layer was dried over MgSO₄, filtered andconcentrated. The crude material was used for next step without furtherpurification. R_(f): 0.44 (10% EtOAC in Hexane). Molecular weight forC₃₇H₇₀NO (M+H)⁺ Calc. 544.55; Found 544.2.

The aminooxy compound was dissolved in EtOH (30 mL), and4-(dimethylamino)butan-2-one (Matrix Scientific, 500 mg, 4.34 mmol) andacetic acid (0.040 mL, 0.70 mmol) was added to the solution. Thereaction mixture was stirred at room temperature for 14 hours. Afteraddition of Et₃N (0.5 mL), the reaction mixture was evaporated. Theresidue was extracted with Et₂O and saturated NaHCO₃ aq., and theorganic layer was dried over MgSO₄, filtered and concentrated. The crudewas purified by silica gel column chromatography (Hexane:EtOAc=1:1) togive compound 5 as a mixture of E/Z-isomers (1.90 g, 2.96 mmol, 85%, 2steps, R_(f)=0.39, 0.21 developed with Hexane:EtOAc=1:1). Molecularweight for C₄₃H₈₁N₂O (M+H)⁺ Calc. 641.63; Found 641.5.

Compound 6 ALNY-202

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 3-dimethylamino-1-propanol (2.37 mL, 23.6 mmol) wasadded dropwise. The resulting mixture was stirred at room temperatureovernight. Taken up 50 mL of the solution, Et₃N (0.822 mL, 5.90 mmol)and ALN-SAN-30 (2.07 g, 3.93 mmol) were added and the reaction mixturewas stirred at room temperature overnight. The reaction mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. The organiclayer was dried over anhydrous MgSO₄, filtered and concentrated. Thecrude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 6. Molecular weight for C₄₂H₇₉N₂O₂ (M+H)⁺ Calc.643.61; Found 643.5.

Compounds of the present invention can be further synthesized by theprocedures described in the following papers, which are herebyincorporated by their entirety:

-   1. Schlueter, Urs; Lu, Jun; Fraser-Reid, Bert. Synthetic Approaches    To Heavily Lipidated Phosphoglyceroinositides. Organic Letters    (2003), 5(3), 255-257-   2. King, J. F.; Allbutt, A. D. Can. J. Chem. 1970, 48, 1754-1769-   3. Mach, Mateusz; Schlueter, Urs; Mathew, Felix; Fraser-Reid, Bert;    Hazen, Kevin C. Comparing n-pentenyl orthoesters and n-pentenyl    glycosides as alternative glycosyl donors. Tetrahedron (2002),    58(36), 7345-7354.

Example 79 Determination of Efficacy of Lipid Particle FormulationsContaining Various Cationic Lipids Using an In Vivo Rodent Factor VIISilencing Model

Factor VII (FVII), a prominent protein in the coagulation cascade, issynthesized in the liver (hepatocytes) and secreted into the plasma.FVII levels in plasma can be determined by a simple, plate-basedcolorimetric assay. As such, FVII represents a convenient model fordetermining sirna-mediated downregulation of hepatocyte-derivedproteins, as well as monitoring plasma concentrations and tissuedistribution of the nucleic acid lipid particles and siRNA.

SEQ Duplex Sequence 5′-3′ ID NO: Target AD-1661GGAfUfCAfUfCfUfCAAGfUfCfUfU FVII AfCdTsdT GfUAAGAfCfUfUGAGAfUGAfUfCfCdTsdT

-   -   Lower case is 2′OMe modification and Nf is a 2′F modified        nucleobase, dT is deoxythymidine, s is phosphothioate        The following cationic lipids were tested:

Com- Molecular pound Compound Structure data A

C₄₂H₇₇N₃O Mol Wt: 640.08  B

C₄₂H₇₈N₂O₂ Mol Wt: 643.08  C

C₄₁H₇₇NS₂ Mol Wt: 648.19  D

C₄₁H₇₇N₃ Mol Wt: 612.07  E

C₄₃H₈₀N₂O₂ Mol Wt: 657.11  F

C₄₃H₈₀N₂O₂ Mol Wt: 657.11  G

C₄₄H₈₂N₂O₂ Mol Wt: 671.134 H

C₄₃H₈₀N₂O Mol Wt: 641.108 I

C₄₃H₈₀N₂O Mol Wt: 641.11  J

C₄₂H₇₈N₂O₂ Mol Wt: 643.081 K

C₄₃H₈₀N₂O₂ Mol Wt: 657.107The cationic lipids shown above were used to formulate liposomescontaining the AD-1661duplex using an in-line mixing method, asdescribed in U.S. provisional patent application 61/228,373. Lipidparticles were formulated using the following molar ratio: 50% Cationiclipid/10% distearoylphosphatidylcholine (DSPC)/38.5% Cholesterol/1.5%PEG-DMG (1-(monomethoxy-polyethyleneglycol)-2,3-dimyristoylglycerol,with an average PEG molecular weight of 2000).

C57BL/6 mice (Charles River Labs, MA) received either saline orformulated siRNA via tail vein injection. At various time points afteradministration, serum samples were collected by retroorbital bleed.Serum levels of Factor VII protein were determined in samples using achromogenic assay (Biophen FVII, Aniara Corporation, OH). To determineliver mRNA levels of Factor VII, animals were sacrificed and livers wereharvested and snap frozen in liquid nitrogen. Tissue lysates wereprepared from the frozen tissues and liver mRNA levels of Factor VIIwere quantified using a branched DNA assay (QuantiGene Assay, Panomics,Calif.).

FVII activity was evaluated in FVII siRNA-treated animals at 48 hoursafter intravenous (bolus) injection in C57BL/6 mice. FVII was measuredusing a commercially available kit for determining protein levels inserum or tissue, following the manufacturer's instructions at amicroplate scale. FVII reduction was determined against untreatedcontrol mice, and the results were expressed as % Residual FVII. Twodose levels (0.05 and 0.005 mg/kg FVII siRNA) were used in the screen ofeach novel liposome composition. FIG. 6 shows a graph illustrating therelative FVII protein levels in animals administered with 0.05 or 0.005mg/kg of lipid particles containing different cationic lipids.

Example 80 siRNA Formulation Using Preformed Vesicles

Cationic lipid containing particles were made using the preformedvesicle method. Cationic lipid, DSPC, cholesterol and PEG-lipid weresolubilised in ethanol at a molar ratio of 40/10/40/10, respectively.The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4)with mixing to a final ethanol and lipid concentration of 30% (vol/vol)and 6.1 mg/mL respectively and allowed to equilibrate at roomtemperature for 2 min before extrusion. The hydrated lipids wereextruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22°C. using a Lipex Extruder (Northern Lipids, Vancouver, BC) until avesicle diameter of 70-90 nm, as determined by Nicomp analysis, wasobtained. This generally required 1-3 passes. For some cationic lipidmixtures which did not form small vesicles hydrating the lipid mixturewith a lower pH buffer (50 mM citrate, pH 3) to protonate the phosphategroup on the DSPC headgroup helped form stable 70-90 nm vesicles.

The FVII siRNA (solubilised in a 50 mM citrate, pH 4 aqueous solutioncontaining 30% ethanol) was added to the vesicles, pre-equilibrated to35° C., at a rate of ˜5 mL/min with mixing. After a final targetsiRNA/lipid ratio of 0.06 (wt/wt) was achieved, the mixture wasincubated for a further 30 min at 35° C. to allow vesiclere-organization and encapsulation of the FVII siRNA. The ethanol wasthen removed and the external buffer replaced with PBS (155 mM NaCl, 3mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by either dialysis or tangential flowdiafiltration. The final encapsulated siRNA-to-lipid ratio wasdetermined after removal of unencapsulated siRNA using size-exclusionspin columns or ion exchange spin columns

Example 81 In Vivo Determination of Efficacy of Novel Lipid Formulations

Test formulations were initially assessed for their FVII knockdown infemale 7-9 week old, 15-25 g, female C57B1/6 mice at 0.1, 0.3, 1.0 and5.0 mg/kg with 3 mice per treatment group. All studies included animalsreceiving either phosphate-buffered saline (PBS, Control group) or abenchmark formulation. Formulations were diluted to the appropriateconcentration in PBS immediately prior to testing. Mice were weighed andthe appropriate dosing volumes calculated (10 μl/g body weight). Testand benchmark formulations as well as PBS (for Control animals) wereadministered intravenously via the lateral tail vein. Animals wereanesthetised 24 h later with an intraperitoneal injection ofKetamine/Xylazine and 500-700 μl of blood was collected by cardiacpuncture into serum separator tubes (BD Microtainer). Blood wascentrifuged at 2,000×g for 10 min at 15° C. and serum was collected andstored at −70° C. until analysis. Serum samples were thawed at 37° C.for 30 min, diluted in PBS and aliquoted into 96-well assay plates.Factor VII levels were assessed using a chromogenic assay (Biophen FVIIkit, Hyphen BioMed) according to manufacturer's instructions andabsorbance measured in microplate reader equipped with a 405 nmwavelength filter. Plasma FVII levels were quantified and ED50s (doseresulting in a 50% reduction in plasma FVII levels compared to controlanimals) calculated using a standard curve generated from a pooledsample of serum from Control animals. Those formulations of interestshowing high levels of FVII knockdown (ED50<<0.1 mg/kg) were re-testedin independent studies at a lower dose range to confirm potency andestablish ED50. The ED50 values of a representative number of compoundsis shown in Table 8

Example 82 Determination of pKa of Formulated Lipids

The pKa's of the different ionisable cationic lipids were determinedessentially as described (Eastman et al 1992 Biochemistry 31:4262-4268)using the fluorescent probe 2-(p-toluidino)-6-naphthalenesulfonic acid(TNS), which is non-fluorescent in water but becomes appreciablyfluorescent when bound to membranes. Vesicles composed of cationiclipid/DSPC/CH/PEG-c-DOMG (40:10:40:10 mole ratio) were diluted to 0.1 mMin buffers (130 mM NaCl, 10 mM CH₃COONH₄, 10 mM MES, 10 mM HEPES) ofvarious pH's, ranging from 2 to 11. An aliquot of the TNS aqueoussolution (1 μM final) was added to the diluted vesicles and after a 30second equilibration period the fluorescent of the TNS-containingsolution was measured at excitation and emission wavelengths of 321 nmand 445 nm, respectively. The pKa of the cationic lipid-containingvesicles was determined by plotting the measured fluorescence againstthe pH of the solutions and fitting the data to a Sigmodial curve usingthe commercial graphing program Igor Pro. The pKa values for arepresentative number of compounds is shown in Table 8

Compound Structure ED50 pKa ALNY-104

2.5 5.65 ALNY-105

1.5 5.60 ALNY-106

0.3 6.85 ALNY-100

0.3 6.4 ALNY-101

0.1 6.43 ALNY-102

2.0 7.3 ALNY-103

2.5 6.98 ALNY-107

0.25 6.63 ALNY-108

0.75 6.55 ALNY-109

2.0 6.75 ALNY-110

2.0 6.5 ALNY-115

1.0 ALNY-116

1.0 ALNY-121

0.5 6.60 ALNY-122

0.55 ALNY-169

2.60 ALNY-144

0.60 ALNY-151

>5.00 5.50 ALNY-152

0.15 6.60 ALNY-156

<0.1 6.08 ALNY-158

1.40 ALNY-190

0.47 6.49 ALNY-192

2.1 7.21 ALNY-200

>5.00 7.57 ALNY-202

0.12 6.52 ALNY-203

5.0 7.07 ALNY-175

2.7 ALNY-149

0.1 5.81 ALNY-160

2.00 5.18 ALNY-201

>5.0 8.02 ALNY-141

0.14 6.62 ALNY-181

0.25 ALNY-140

>5.0 4.95 ALNY-148

0.3 6.53 ALNY-117

>5.0

Example 83 Synthesis of Pyridine Containing Lipids

Compound 7006: To a stirred solution of 3-cyclopentene-1-ol (7005, 2.0g, 23.77 mmol, 1.0 eq) in dichloromethane, was added imidazole (3.88 g,57.08 mmol, 2.4 eq) portion wise, followed by TBDPSCl (6.1 mL, 23.77mmol, 1.0 eq) drop wise at room temperature under argon atmosphere. Thereaction was continued at room temperature for overnight. Aftercompletion of the reaction was washed with water, brine, combinedorganic layers were dried on MgSO₄, evaporated the solvent under reducedpressure and purified by column chromatography using 3% ether in hexaneas a gradients to get pure TBDPS protected 3-cyclopentenol (7006) as oilan 87% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (dd, J=7.8, 1.4, 4H),7.53-7.31 (m, 6H), 5.60 (d, J=7.0, 2H), 4.55 (dq, J=6.6, 4.3, 1H),2.57-2.24 (m, 4H), 1.05 (s, 9H). Calc. mass for C₂₁H₂₆OSi is 322.0;found 322.5

Compound 7007: To a cooled solution of OsO₄ (0.031 g, 0.12 mmol, 0.01eq), NMO (50% in water, 5.14 mL, 24.84 mmol, 2.0 eq) in water wastreated drop wise with a solution of compound 7006 (4.0 g, 12.42 mmol,1.0 eq) in acetone. The reaction was continued at room temperature forovernight, after complete consumption of the starting material, solventwas evaporated and the reaction mixture was extracted into ethyl acetate(2×). Combined organics were dried over Na₂SO₄, solvent was evaporated,and the mixture was purified by column chromatography using hexane andethyl acetate (50%) as gradients to get pure dihydroxylated compound(7007) as an oil in 92% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.70-7.53 (m,4H), 7.49-7.28 (m, 6H), 4.53-4.37 (m, 1H), 4.29 (dd, J=8.0, 4.6, 2H),2.08 (d, J=4.0, 1H), 1.99-1.78 (m, 4H), 1.58 (s, 1H), 1.04 (d, J=10.2,9H). Calc. mass for C21H₂₈O₃Si is 356.5; found 379.0 (+Na).

Compound 7008: A mixture of dihydroxylated compound (7007, 4.0 g, 11.4mmol, 1.0 eq), dilinoleyl ketone (6.0 g, 11.4 mmol, 1.0 eq) andp-toluenesulfonic acid (0.21 g, 1.14 mmol, 0.1 eq) in toluene wasrefluxed under Dean-stock conditions for 4 hours. TLC shows completionof the reaction, solvent was evaporated and directly purified on columnchromatography using hexane and ethyl acetate (5%) as gradients to get95% of the pure ketal (7008) as an oil. ¹H NMR (400 MHz, CDCl₃) δ7.75-7.57 (m, 4H), 7.49-7.28 (m, 6H), 5.48-5.16 (m, 8H), 4.62-4.35 (m,3H), 2.77 (dd, J=14.0, 6.8, 4H), 2.12-1.94 (m, 10H), 1.59 (dd, J=15.4,6.4, 2H), 1.48-1.15 (m, 35H), 1.07 (d, J=22.0, 14H), 0.88 (t, J=6.8,6H). Calc. mass for C₅₈H₉₂O₃Si is 864.5; found 887.5 (+Na).

Compound 7009: To a stirred solution of compound 7008 (3.94 g, 4.55mmol, 1.0 eq) in dichloromethane was added drop wise a solution of 1MTBAF (22.8 mL, 22.79 mmol, 5.0 eq) in THF at 0° C., and the reaction wascontinued at room temperature until completion of the reaction.Concentrated the reaction, mixture was directly loaded onto the columnand purified by column using hexane and ethyl acetate (20%) as gradientsto get pure product (7009) as an oil in 87% yield. ¹H NMR (400 MHz,CDCl₃) δ 5.53-5.23 (m, 8H), 4.71-4.61 (m, 2H), 4.56 (s, 1H), 2.80 (t,J=6.4, 4H), 2.41 (dd, J=8.5, 6.7, 2H), 2.23 (dd, J=13.9, 6.0, 2H), 2.07(q, J=6.8, 8H), 1.70-1.48 (m, 6H), 1.39 (dddd, J=23.0, 20.3, 11.9, 6.6,36H), 0.92 (q, J=7.2, 6H). Calc. mass for C₄₂H₇₄O₃ is 627.0; found627.5.

Compound 7301: To a solution of the hydroxy compound (7009, 2.5 g, 4.0mmol, 1.0 eq) in dichloromethane was added PCC (1.3 g, 6.0 mmol, 1.5 eq)adsorbed on silica gel portion wise at room temperature and continuedthe stirring until the completion of the reaction. Reaction mixture wasfiltered through the celite, washed with the dichloromethane, thecombined organics were washed with water, separated the organic layer,dried, concentrated and purified the mixture using hexane and ethylacetate (20%) as gradients to get pure oil material of the compound 7301in 68% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.56-5.22 (m, 8H), 5.05-4.78 (m,2H), 2.80 (t, J=6.4, 4H), 2.58-2.49 (m, 3H), 2.07 (q, J=6.8, 8H),1.72-1.50 (m, 4H), 1.47-1.21 (m, 36H), 1.09 (s, 1H), 0.91 (t, J=6.8,6H). Calc. mass for C₄₂H₇₂O₃ is 625.0; found 625.5.

Compound 7303 (ALNY-137): NaBH(OAc)₃ (0.16 g, 0.75 mmol, 1.5 eq) wasadded portion wise to a solution of N-methyl-3-pyridyl methylamine 7302(0.06 g, 0.5 mmol, 1.0 eq) in dichloromethane at room temperature. After10 minutes of stirring, a solution of ketone derivative (7301, 0.34 g,0.55 mmol, 1.1 eq) in dichloromethane was added drop wise at roomtemperature and continued the reaction for overnight. After completionof the reaction, mixture was quenched with 1N NaHCO₃, extracted withdichloromethane; combined organic layers were washed with brine anddried on MgSO₄. The concentrated mixture was purified by columnchromatography using dichloromethane, methanol (5%), triethylamine(0.3%) as gradients to get pure product (7303) in 0.18 g (51%)quantities. ¹H NMR (400 MHz, CDCl₃) δ 8.63-8.32 (m, 2H), 7.66 (d, J=7.8,1H), 7.24 (dd, J=7.3, 4.4, 1H), 5.48-5.17 (m, 8H), 4.53 (dd, J=10.0,5.5, 2H), 3.51 (s, 2H), 2.93-2.61 (m, 5H), 2.24 (dt, J=12.6, 6.1, 2H),2.13 (s, 3H), 2.04 (q, J=6.7, 8H), 1.71 (ddd, J=15.9, 10.2, 4.6, 4H),1.52 (d, J=7.3, 2H), 1.46-1.16 (m, 36H), 0.88 (t, J=6.7, 6H). ¹³C NMR(101 MHz, cdcl₃) δ 150.47, 148.80, 136.76, 134.74, 130.39, 130.38,130.35, 128.16, 128.14, 123.56, 116.10, 78.93, 77.54, 77.23, 76.91,64.01, 57.35, 39.32, 37.24, 37.01, 35.85, 31.74, 30.18, 30.10, 29.91,29.88, 29.82, 29.79, 29.70, 29.57, 29.55, 29.52, 27.46, 27.44, 27.41,25.84, 24.58, 23.80, 22.80, 14.31. Calc. mass for the C₄₉H₈₂N₂O₂: 730.6;found 731.5.

Compound 7305 (ALNY-136): Prepared by similar experimental conditionsused as for compound 7303, using NaBH(OAc)₃ (0.2 g, 0.98 mmol, 1.5 eq),N-methylpyridine derivative 7304 (0.09 g, 0.65 mmol, 1.0 eq) and ketonederivative (7301, 0.45 g, 0.72 mmol, 1.1 eq), this gave 0.3 g (62%) ofthe pure product 7305. ¹H NMR (400 MHz, CDCl₃) δ 8.49 (dd, J=4.5, 1.5,2H), 7.11 (d, J=5.9, 2H), 5.45-5.22 (m, 8H), 4.51 (t, J=4.5, 2H),2.95-2.56 (m, 9H), 2.31 (s, 3H), 2.19 (dt, J=12.5, 6.2, 2H), 2.04 (q,J=6.7, 8H), 1.71-1.58 (m, 4H), 1.50 (d, J=6.9, 2H), 1.32 (ddd, J=22.2,13.8, 7.6, 36H), 0.88 (t, J=6.8, 6H). ¹³C NMR (101 MHz, CDCl₃) δ 149.94,149.58, 130.39, 130.34, 128.16, 128.13, 128.11, 124.31, 116.12, 78.84,77.54, 77.22, 76.90, 63.88, 56.34, 39.43, 37.22, 37.04, 35.94, 33.10,31.73, 30.17, 30.08, 29.91, 29.87, 29.81, 29.78, 29.69, 29.56, 29.54,29.51, 27.46, 27.43, 27.41, 25.83, 24.55, 23.74, 22.79, 14.30. Calc.mass for the C₅₀H₈₄N₂O₂: 744.65; found 745.5.

Example 84 Synthesis of Amide Linked Lipid

To a stirred suspension of N,N-dimethylamino propionic acidhydrochloride (1, 0.198 g, 1.3 mmol, 1.0 eq) in DCM was added HBTU (0.59g, 1.56 mmol, 1.2 eq) and DIPEA (0.71 mL, 3.9 mmol, 3.0 eq) at roomtemperature. After stirred for 10 minutes, a solution of amine (2, 0.7g. 1.3 mmol, 1.0 eq) in DCM was added drop wise at room temperature andcontinued the stirring until completion of the reaction. Reactionmixture was diluted with DCM, washed with saturated NaHCO₃ solutionfollowed by brine, organic layer was separated and dried over MgSO₄,concentrated and purified by the silica gel column chromatography usingDCM:MeOH (5%) as gradients to get pure oily compound 3 (ALNY-201) in 70%yield.

¹H NMR (400 MHz, CDCl₃) δ 7.18 (brs, 1H), 5.47-5.19 (m, 8H), 3.18-3.07(m, 4H), 2.76 (t, J=6.5, 4H), 2.70 (s, 6H), 2.60 (t, J=6.0, 2H), 2.04(q, J=6.8, 9H), 1.48 (brs, 1H), 1.40-1.14 (m, 43H), 0.88 (t, J=6.8, 6H).¹³C NMR (101 MHz, CDCl₃) δ 172.26, 130.41, 130.36, 128.17, 128.15,77.54, 77.22, 76.90, 55.70, 43.85, 43.02, 37.90, 31.99, 31.74, 30.25,29.92, 29.86, 29.81, 29.57, 27.47, 27.42, 26.84, 25.85, 22.79, 14.29.Calc. mass for the C43H80N2O: 640.6; found 641.5.

Example 85 Synthesis of Carbamate and Urea Linked Lipids Compound 1033

Stage-1:

Chemicals/Reagents & S. No solvents M. Wt. Mol Eq. Qty. 1 Alcohol 1030528 0.095 1 50 g 2 DCM 500 ml 3 Triethylamine (TEA) 101.2 0.378 4 53 ml4 DMAP 122.17 0.0095 0.1 1.2 g 5 Mesyl chloride 114.55 0.19 2 15 ml

To a solution of Alcohol 1030 in DCM (400 ml) under Ar atmosphere, wasadded TEA and DMAP and stirred at room temperature under Ar atmosphere.Reaction mass was cooled to −5° C. and the solution of mesyl chloride inDCM (100 ml) was added slowly at temperature below −5° C. and allowed towarm to RT after addition. After 30 minutes (TLC), reaction mass wasquenched with ice cold water (20 ml). Organic layer was separated,washed with 1N HCl (30 ml), water, brine, dried over sodium sulfate andevaporated at reduced pressure to obtain pure product 1031 (55 g, yield95.5%) as an yellow liquid. HPLC: 99.8%; ¹H NMR (400 MHz, CDCl₃): δ 0.89(t, 6H, J=6.8 Hz), 1.2-1.5 (m, 36H), 1.67 (m, 4H), 2.05 (q, 8H, J=6.8Hz), 2.77 (t, 4H, J=6.4 Hz), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m,8H). ¹³CNMR (100 MHz, CDCl₃): δ 14.0, 22.5, 24.9, 25.6, 27.2, 29.2,29.3, 29.4, 29.5, 29.6, 31.5, 34.4, 38.6, 45.9, 84.3, 127.9, 128.0,130.0, 130.1.

Stage-2:

Chemicals/Reagents & S. No solvents M. Wt. Mol. Eq. Qty. 1 Mesylate 1031606 0.0165 1 10 g 2 Dimethylformamide (DMF) 100 ml 3 Sodium cyanide 490.0330 2 1.617 g

To a solution of sodium cyanide in DMF under Ar atmosphere, was addedstage-1 product in DMF slowly and then heated to 55° C. for 24 hrs(HPLC). It was then cooled to room temperature, diluted with water andextracted with ethyl acetate (several times). The combined organic layerwas washed with water, brine, dried over sodium sulfate and evaporatedat reduced pressure to obtain crude product, which was purified silicagel chromatography using 1% ether/hexane as eluent to afford pureproduct 1032 (5.8 g, yield: (62%) as a pale yellow liquid. ¹H NMR (400MHz, CDCl₃): δ 0.87 (t, 6H, J=6.8 Hz), 1.25 (m, 38H), 1.52 (m, 4H), 2.03(q, 8H, J=6.8 Hz), 2.47 (m, 1H), 2.76 (t, 4H, J=6.4 Hz), 5.32 (m, 8H).

Stage-3:

Chemicals/Reagents & S. No solvents M. Wt. Mol. Eq. Qty. 1 Nitrile 1032538 0.0097 1 5.2 g 2 Lithium aluminiumhydride 38 0.0387 4 1.5 g 3Tetrahydrofuran (THF) 52 ml

To a suspension of lithium aluminiumhydride in dry THF at Ar atmosphere,was added stage-2 product in THF at 0° C. drop-wise. It was then allowedto warm to room temperature (RT) and stirred for 20 hrs at RT (TLC). Itwas cooled to 0° C. and quenched with saturated solution of sodiumsulfate. The quenched mass was filtered through celite bed and washedwith ethyl acetate. The combined filtrate was evaporated at reducedpressure to obtain crude product, which was purified by silica gelchromatography using 10% ethyl acetate in hexane to afford pure product1033 (3.7 g, yield: 71%) as pale brown liquid, HPLC: 93.8%. ¹H NMR (400MHz, CDCl₃): δ 0.87 (t, 6H, J=6.8 Hz), 1.27 (m, 48H), 2.03 (q, 8H, J=6.8Hz), 2.60 (d, 2H, J=4.0 Hz), 2.76 (t, 4H, J=6.4 Hz), 5.31 (m, 8H).¹³CNMR (100 MHz, CDCl₃): δ 14.1, 22.6, 25.6, 26.8, 27.1, 27.2, 29.3,29.5, 29.6, 30.1, 31.5, 40.9, 45.2, 128.0, 130.1. LC-MS: 543 (M+).

Compound 1003 (ALNY-192)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 3-dimethylamino-1-propanol (1001, 2.43 g, 23.6 mmol)was added dropwise. The resulting mixture was stirred at roomtemperature overnight. Taken up 50 mL of the solution, Et₃N (0.822 mL,5.90 mmol) and ALN-SAN-30 (2.08 g, 3.93 mmol) were added and thereaction mixture was stirred at room temperature overnight. The reactionmixture was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. Theorganic layer was dried over anhydrous MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 1003 (1.66 g, 2.53 mmol, 64%, R_(f)=0.22 with5% MeOH in CH₂Cl₂). ¹H NMR (CDCl₃, 400 MHz) 5.30-5.41 (m, 8H), 4.37 (d,J=8.0 Hz, 1H), 4.09 (t, J=6.0 Hz, 2H), 3.57 (brs, 1H), 2.78 (t, J=6.0Hz, 4H), 2.33 (t, J=8.0 Hz, 2H), 2.23 (s, 6H), 2.02-2.06 (m, 8H),1.76-1.80 (m, 2H), 1.27-1.45 (m, 40H), 0.89 (t, J=8.0 Hz, 6H). ¹³C NMR(CDCl₃, 100 MHz) 156.5, 130.4, 130.3, 128.2, 128.1, 63.2, 56.6, 51.4,45.7, 35.7, 31.7, 29.9, 29.8, 29.7, 29.6, 29.5, 27.7, 27.5, 27.4, 26.0,25.8, 22.8, 14.3. Molecular weight for C₄₃H₈₁N₂O₂ (M+H)⁺ Calc. 657.63;Found 657.5.

Compound 1004 (ALNY-200)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 3-dimethylamino-1-propanol (1001, 2.43 g, 23.6 mmol)was added dropwise. The resulting mixture was stirred at roomtemperature overnight. Taken up 50 mL of the solution, Et₃N (0.697 mL,5.00 mmol) and amine 1033 (1.71 g, 3.15 mmol) were added and thereaction mixture was stirred at room temperature overnight. The reactionmixture was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. Theorganic layer was dried over anhydrous MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 1004 (1.14 g, 1.70 mmol, 54%, R_(f)=0.13 with5% MeOH in CH₂Cl₂). Molecular weight for C₄₄H₈₃N₂O₂ (M+H)⁺ Calc. 671.65;Found 671.5.

Compound 1007 (ALNY-202)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 2-dimethylaminoethanol (1005, 2.37 mL, 23.6 mmol) wasadded dropwise. The resulting mixture was stirred at room temperatureovernight. Taken up 50 mL of the solution, Et₃N (0.822 mL, 5.90 mmol)and ALN-SAN-30 (2.07 g, 3.92 mmol) were added and the reaction mixturewas stirred at room temperature overnight. The reaction mixture wasdiluted with CH₂Cl₂ and washed with saturated NaHCO₃ aq. The organiclayer was dried over anhydrous MgSO₄, filtered and concentrated. Thecrude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 1007 (1.78 g, 2.77 mmol, 71%, 2 steps,R_(f)=0.26 developed with 5% MeOH in CH₂Cl₂). Molecular weight forC₄₂H₇₉N₂O₂ (M+H)⁺ Calc. 643.61; Found 643.5.

Compound 1008 (ALNY-203)

To a solution of N,N′-disuccinimidyl carbonate (5.50 g, 21.5 mmol) inCH₂Cl₂ (200 mL), 2-dimethylaminoethanol (1005, 2.37 mL, 23.6 mmol) wasadded dropwise. The resulting mixture was stirred at room temperatureovernight. Taken up 50 mL of the solution, Et₃N (0.697 mL, 5.00 mmol)and 1033 (440 mg, 0.812 mmol) were added and the reaction mixture wasstirred at room temperature overnight. The reaction mixture was dilutedwith CH₂Cl₂ and washed with saturated NaHCO₃ aq. The organic layer wasdried over anhydrous MgSO₄, filtered and concentrated. The crude waspurified by silica gel column chromatography (0-5% MeOH in CH₂Cl₂) togive compound 8 (332 mg, 0.505 mmol, 62%, R_(f)=0.30 with 5% MeOH inCH₂Cl₂). Molecular weight for C₄₃H₈₁N₂O₂ (M+H)⁺ Calc. 657.63; Found657.5.

Example 86 Synthesis of Guanidinium Linked Lipids Guanidinium AnalogsSynthesis of 2064

Synthesis of 2063: To a solution of 2058 (6.7 g, 0.0112 mol) inDMF/Ethyl acetate mixture was added Bis-Boc-S-methylisothiourea (3.4 g,0.0118 mol) and triethylamine (3.5 mL, 0.246 mol) at 0° C. To thehomogeneous solution was added HgCl₂ (3.3 g, 0.0123 mol) at 0° C. andstirred at RT for 1 hr. TLC showed the absence of starting material. Thereaction mass was then diluted with ethyl acetate (100 ml). Filteredthrough a pad of celite and washed with ethyl acetate. The filtrate wasgiven water wash (2×150 ml) and brine wash (200 ml). The hazy organiclayer was again filtered through a pad of celite/230-400 mesh silicagel/celite. The filtrate was evaporated at reduced pressure to obtainthe crude product, which was purified by neutral alumina chromatographyusing and DCM/Hexane as eluent. The product got eluted at 40% DCM inHexane as yellow liquid (Yield 5.2 g, 55%). ¹H NMR (400 MHz, CDCl₃):0.89 (t, 6H, J=6.8 Hz), 1.27-1.46 (m, 43H), 1.49 (s, 9H), 1.50 (s, 9H),2.02 (q, 8H, J₁=6.8 Hz, J₂=6.8 Hz), 2.12 (d, 2H, J=7.2 Hz), 2.16 (s,3H), 2.46 (t, 2H, J=5.6 Hz), 2.77 (t, 4H, J=6 Hz), 3.47 (m, 2H), 5.30(m, 8H), 8.67 (s, 1H), 11.48 (9s, 1H).

Synthesis of 2064 (ALNY-139): To a solution of 2063 (5.2 g, 0.0062 mol)in 10 ml of DCM at 0° C., was added 10 ml of TFA in 60 ml of DCM slowly.After addition the reaction mass was stirred at RT for 3 hrs. The TLCshowed the absence of starting material. Excess TFA was removed undervacuum, to obtain the required product as brown viscous liquid (5.3 g,78%). ¹H NMR (400 MHz, CDCl₃): 0.89 (t, 6H, J=6.8 Hz), 1.27-1.46 (m,44H), 1.78 (s, 1H), 2.02 (q, 8H, J₁=6.4 Hz, J₂=6.8 Hz), 2.77 (t, 4H,J=6.4 Hz), 2.86 (s, 3H), 2.92-3.01 (m, 2H), 3.27-3.39 (m, 2H), 3.76-3.9(m, 2H), 5.30 (m, 8H), 7.12 (m, 2H), 8.41 (m, 1H), 10.02 (m, 3H). ¹³CNMR (100 MHz, CDCl₃): 14.0, 22.5, 25.6, 25.8, 26.0, 27.17, 27.19, 27.6,29.3, 29.33, 29.5, 29.6, 31.0, 31.5, 33.9, 36.3, 41.0, 54.1, 55.2, 62.0,62.19, 111.4, 114.3, 117.1, 119.9, 127.9, 127.95, 130.1, 130.2, 152.1,155.0, 157.4, 161.2, 161.6, 161.96, 162.3. MS: 1093 (tetra TFA salt).

Preparation of Compound 7204

Preparation of Compound 7013: To a mixture of 1,2,4-butanetriol (7012,21.2 g, 200 mmol, 5.0 eq), dilinoleyl ketone (21.0 g, 40.0 mmol, 1.0 eq)and p-toluenesulfonic acid (0.76 g, 4.0 mmol, 0.1 eq) in toluene wasrefluxed under Dean-stock conditions for overnight. After completion ofthe reaction, was cooled, evaporated the solvent and purified by columnchromatography using hexane and ethyl acetate (15%) as gradients gavedesired ketal (7013) in 47% yield as an oil. ¹H NMR (400 MHz, CDCl₃) δ5.48-5.24 (m, 8H), 4.32-4.17 (m, 1H), 4.08 (dd, J=7.8, 6.1, 1H),3.86-3.74 (m, 2H), 3.53 (t, J=8.0, 1H), 2.77 (t, J=6.4, 4H), 2.30-2.19(m, 1H), 2.05 (q, J=6.8, 8H), 1.88-1.75 (m, 2H), 1.69-1.51 (m, 4H),1.42-1.19 (m, 36H), 0.89 (t, J=6.8, 6H). Calc. mass for C₄₁H₇₄O₃ is614.5; found 637.3 (+Na).

Synthesis of compound 7201: To a solution of compound 7013 (11.6 g, 18.9mmol, 1.0 eq) and triethyl amine (5.45 mL, 37.7 mmol, 2.0 eq) indichloromethane at 0° C. was added drop wise a solution ofmethanesulfonyl chloride (1.74 mL, 22.67 mmol, 1.2 eq), and the reactionwas continued at room temperature for 1 h. After completion of thereaction, was washed with water, brine, and combined organics were driedon MgSO₄. The concentrated mixture was purified on column chromatographyusing hexane and ethyl acetate (20%) as gradients to get pure mesylatedderivative (7201) as an oil in 93% yield. ¹H NMR (400 MHz, CDCl₃) δ5.48-5.22 (m, 8H), 4.35 (qd, J=10.0, 4.9, 2H), 4.25-4.14 (m, 1H),4.13-4.03 (m, 1H), 3.53 (t, J=7.6, 1H), 3.02 (s, 3H), 2.77 (t, J=6.4,4H), 2.13-1.85 (m, 10H), 1.57 (dd, J=18.2, 9.2, 4H), 1.44-1.15 (m, 36H),0.89 (t, J=6.7, 6H). Calc. mass for C₄₂H₇₆O₅S is 693.1; found 693.2.

Synthesis of compound 7202: To a solution of the compound 7201 (2.0 g,3.0 mmol, 1.0 eq) in DMF was added solid NaN₃ (0.98 g, 15.0 mmol, 5.0eq) at room temperature and the reaction was continued at 65° C. untilthe completion of the reaction. Reaction mixture was poured onto icewater, extracted into ethyl acetate, combined organics were dried onNa₂SO₄, concentrated, purified on column chromatography using hexane andethyl acetate (5%) as gradients to get pure azido (7202) derivative in89% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.53-5.19 (m, 8H), 4.21-3.97 (m,2H), 3.57-3.29 (m, 3H), 2.76 (t, J=6.4, 4H), 2.04 (q, J=6.8, 8H), 1.80(m, 2H), 1.66-1.43 (m, 4H), 1.40-1.07 (m, 36H), 0.88 (t, J=6.8, 6H).Calc. mass for C₄₁H₇₃N₃O₂ is 640.0; found 612.5 (—N₂).

Synthesis of compound 7203: To a solution of compound 7202 (1.7 g, 2.65mmol, 1.0 eq) in anhydrous tetrahydrofuran, was added drop wise a 1Msolution of LAH (3.98 mL, 3.98 mmol, 1.5 eq) at 0° C. Reaction wascontinued at room temperature, after completion of the reaction wasquenched with saturated solution of Na₂SO₄ slowly at 0° C. Compound wasextracted into excess amount of ethyl acetate, organic layer was washedwith brine, dried over Na₂SO₄, concentrated and further dried on vacuumto get pure amine (7203) in 90% yield, and this has been used directlywithout further purification. ¹H NMR (400 MHz, CDCl₃) δ 5.51-5.16 (m,8H), 4.13 (dd, J=9.3, 3.6, 1H), 4.03 (dd, J=7.5, 6.1, 1H), 3.46 (t,J=7.8, 1H), 2.96-2.67 (m, 6H), 2.20-1.92 (m, 8H), 1.82-1.49 (m, 6H),1.46-1.12 (m, 38H), 0.88 (t, J=6.8, 6H). Calc. mass for the C₄₁H₇₅NO₂ is614.0; found 614.5.

Synthesis of compound 7204 (ALNY-232): To a solution of amine 7203 (0.61g, 1.0 mmol, 1.0 eq) and DIPEA (1.84 mL, 10.0 mmol, 10.0 eq) in asolvent mixture (DCM:DMF) was added 1H-pyrazole-1-carboxamidinehydrochloride (1.46 g, 10.0 mmol, 10.0 eq) portion wise at roomtemperature, under argon atmosphere. The reaction was continued forovernight, after completion of the reaction, was poured onto ice, andextracted with the ethyl acetate. The combined organics were washed withwater, brine, dried over Na₂SO₄ and purified by preparativechromatography to get pure 0.16 g (25%) of the guanidine derivative(7204). 1H NMR (400 MHz, CDCl3) δ 11.76 (s, 1H), 7.99 (t, J=6.3, 1H),7.44 (s, 2H), 5.48-5.20 (m, 8H), 4.24-4.00 (m, 2H), 3.54 (dd, J=7.3,6.2, 1H), 3.32 (d, J=3.0, 2H), 3.09 (dt, J=10.5, 5.3, 1H), 2.76 (t,J=6.5, 4H), 2.03 (q, J=6.8, 8H), 1.90-1.77 (m, 1H), 1.76-1.49 (m, 6H),1.48-1.05 (m, 34H), 0.87 (dd, J=6.8, 6H). ¹³C NMR (101 MHz, cdcl₃) δ158.96, 130.41, 130.36, 130.33, 128.18, 128.14, 113.52, 77.54, 77.22,76.90, 76.60, 72.36, 69.54, 46.09, 38.39, 37.68, 37.01, 34.09, 31.74,30.10, 29.92, 29.78, 29.76, 29.56, 29.55, 29.53, 27.47, 27.46, 27.41,25.84, 24.37, 24.12, 22.79, 14.31, 8.86. Calc. mass for the C₄₂H₇₇N₃O₂is 656.0; found 656.2.

Example 87 Synthesis of Ester Linked Lipids Lipid Discovery EsterAnalogs

Experimental

Compound 7002: Magnesium (711 mg, 29.25 mmol) was placed in a roundbottle flask. THF (30 mL) and 2-3 mg of I₂ were added. The mixture waswarmed at 50° C. and oleylbromide (7001, 6.46 g, 19.50 mmol) was addedslowly. When ˜1 mL of oleylbromide was added, formation of the Grignardreagent was initiated. After addition of the reast of oleylbromide, theGrignard reagent was stirred at room temperature for 60 min then slowlyadded to a solution of 1,1′-carbonyldiimidazole (1.54 g, 9.51 mmol) inTHF (100 mL) at −50° C. The reaction mixture was kept stirring at −50°C. for 30 min then at room temperature for 60 min The reaction wasquenched with 40 mL of saturated NH₄Cl aq. and the mixture was extractedwith Et₂O and H₂O. The organic layer was dried over MgSO₄, filtered andconcentrated. The crude was purified by silica gel column chromatography(0-5% Et₂O in Hexane) to give compound 7002 (2.70 g, 5.09 mmol, 53%,R_(f)=0.48 developed with 5% EtOAc in Hexane). Molecular weight forC₃₇H₇₁O (M+H)⁺ Calc. 531.55; Found 531.5.

Compound 7003: To a solution of compound 7002 (1.36 g, 2.56 mmol) in THF(25 mL), 1 M lithium aluminum hydride in THF (5.12 mL, 5.12 mmol) wasadded at 0° C. The reaction mixture was stirred at room temperature for3 hours. The reaction was quenched with saturated Na₂SO₄ aq. (20 mL),then extracted with Et₂O and H₂O. The organic layer was dried overMgSO₄, filtered and concentrated. The crude was purified by silica gelcolumn chromatography (0-5% Et₂O in Hexane) to give compound 7003 (942mg, 1.77 mmol, 69%, R_(f)=0.26 developed with 5% EtOAc in Hexane).

Compound 7004: To a solution of compound 7003 (940 mg, 1.76 mmol) and4-(dimethylamino)butyric acid hydrochloride (355 mg, 2.12 mmol) inCH₂Cl₂ (15 mL), diisopropylethylamine (0.920 mL, 5.28 mmol),N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (406 mg,2.12 mmol) and DMAP (43 mg, 0.352 mmol) were added. The reaction mixturewas stirred at room temperature for 14 hours. The reaction mixture wasdiluted with CH₂Cl₂ (100 mL) and washed with saturated NaHCO₃ aq. (50mL). The organic layer was dried over MgSO₄, filtered and concentrated.The crude was purified by silica gel column chromatography (0-5% MeOH inCH₂Cl₂) to give compound 7004 (817 mg, 1.26 mmol, 72%, R_(f)=0.29developed with 5% MeOH in CH₂Cl₂). Molecular weight for C₄₃H₈₄NO₂ (M+H)⁺Calc. 646.65; Found 646.5.

Compound 7006: To a stirred solution of 3-cyclopentene-1-ol (7005, 2.0g, 23.77 mmol, 1.0 eq) in dichloromethane, was added imidazole (3.88 g,57.08 mmol, 2.4 eq) portion wise, followed by TBDPSCl (6.1 mL, 23.77mmol, 1.0 eq) drop wise at room temperature under argon atmosphere. Thereaction was continued at room temperature for overnight. Aftercompletion of the reaction was washed with water, brine, combinedorganic layers were dried on MgSO₄, evaporated the solvent under reducedpressure and purified by column chromatography using 3% ether in hexaneas a gradients to get pure TBDPS protected 3-cyclopentenol (7006) as oilan 87% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.67 (dd, J=7.8, 1.4, 4H),7.53-7.31 (m, 6H), 5.60 (d, J=7.0, 2H), 4.55 (dq, J=6.6, 4.3, 1H),2.57-2.24 (m, 4H), 1.05 (s, 9H). Calc. mass for C₂₁H₂₆OSi is 322.0;found 322.5

Compound 7007: To a cooled solution of OsO₄ (0.031 g, 0.12 mmol, 0.01eq), NMO (50% in water, 5.14 mL, 24.84 mmol, 2.0 eq) in water wastreated drop wise with a solution of compound 7006 (4.0 g, 12.42 mmol,1.0 eq) in acetone. The reaction was continued at room temperature forovernight, after complete consumption of the starting material, solventwas evaporated and the reaction mixture was extracted into ethyl acetate(2×). Combined organics were dried over Na₂SO₄, solvent was evaporated,and the mixture was purified by column chromatography using hexane andethyl acetate (50%) as gradients to get pure dihydroxylated compound(7007) as an oil in 92% yield. ¹H NMR (400 MHz, CDCl₃) δ 7.70-7.53 (m,4H), 7.49-7.28 (m, 6H), 4.53-4.37 (m, 1H), 4.29 (dd, J=8.0, 4.6, 2H),2.08 (d, J=4.0, 1H), 1.99-1.78 (m, 4H), 1.58 (s, 1H), 1.04 (d, J=10.2,9H). Calc. mass for C21H₂₈O₃Si is 356.5; found 379.0 (+Na).

Compound 7008: A mixture of dihydroxylated compound (7007, 4.0 g, 11.4mmol, 1.0 eq), dilinoleyl ketone (6.0 g, 11.4 mmol, 1.0 eq) andp-toluenesulfonic acid (0.21 g, 1.14 mmol, 0.1 eq) in toluene wasrefluxed under Dean-stock conditions for 4 hours. TLC shows completionof the reaction, solvent was evaporated and directly purified on columnchromatography using hexane and ethyl acetate (5%) as gradients to get95% of the pure ketal (7008) as an oil. ¹H NMR (400 MHz, CDCl₃) δ7.75-7.57 (m, 4H), 7.49-7.28 (m, 6H), 5.48-5.16 (m, 8H), 4.62-4.35 (m,3H), 2.77 (dd, J=14.0, 6.8, 4H), 2.12-1.94 (m, 10H), 1.59 (dd, J=15.4,6.4, 2H), 1.48-1.15 (m, 35H), 1.07 (d, J=22.0, 14H), 0.88 (t, J=6.8,6H). Calc. mass for C₅₈H₉₂O₃Si is 864.5; found 887.5 (+Na).

Compound 7009: To a stirred solution of compound 7008 (3.94 g, 4.55mmol, 1.0 eq) in dichloromethane was added drop wise a solution of 1MTBAF (22.8 mL, 22.79 mmol, 5.0 eq) in THF at 0° C., and the reaction wascontinued at room temperature until completion of the reaction.Concentrated the reaction, mixture was directly loaded onto the columnand purified by column using hexane and ethyl acetate (20%) as gradientsto get pure product (7009) as an oil in 87% yield. ¹H NMR (400 MHz,CDCl₃) δ 5.53-5.23 (m, 8H), 4.71-4.61 (m, 2H), 4.56 (s, 1H), 2.80 (t,J=6.4, 4H), 2.41 (dd, J=8.5, 6.7, 2H), 2.23 (dd, J=13.9, 6.0, 2H), 2.07(q, J=6.8, 8H), 1.70-1.48 (m, 6H), 1.39 (dddd, J=23.0, 20.3, 11.9, 6.6,36H), 0.92 (q, J=7.2, 6H). Calc. mass for C₄₂H₇₄O₃ is 627.0; found627.5.

Compound 7010 (ALNY-236): To a stirred solution of alcohol 7009 (0.8 g,1.27 mmol, 1.0 eq), N,N-dimethylamino propionic acid hydrochloride (0.23g, 1.53 mmol, 1.2 eq), DIPEA (0.7 mL, 3.81 mmol, 3.0 eq) indichloromethane at room temperature was added EDCI (0.26 g, 1.4 mmol,1.1 eq) followed by DMAP (0.015 g, 0.12 mmol, 0.1 eq) portion wise underargon and continued the reaction for overnight at room temperature.After completion of the reaction was diluted with dichloromethane,washed with saturated solution of NaHCO₃, brine, combined organics weredried over MgSO₄, evaporated the solvent and purified by columnchromatography using dichloromethane: methanol (5%) as gradients to getpure product 7010 in 79% (0.72 g) yields. ¹H NMR (400 MHz, CDCl₃) δ5.47-5.18 (m, 8H), 4.64 (d, J=5.3, 2H), 2.76 (t, J=6.3, 4H), 2.58 (t,J=7.3, 2H), 2.43 (dd, J=9.2, 5.3, 2H), 2.30 (dd, J=14.0, 6.3, 2H), 2.22(s, 6H), 2.04 (q, J=6.7, 8H), 1.72-1.56 (m, 4H), 1.49 (s, 2H), 1.32(ddd, J=22.1, 13.7, 7.6, 37H), 0.88 (t, J=6.8, 6H). ¹³C NMR (101 MHz,CDCl₃) δ 171.86, 130.20, 130.16, 130.13, 130.09, 127.95, 127.89, 127.87,113.27, 78.29, 77.29, 76.97, 76.66, 73.95, 54.68, 45.24, 38.31, 36.25,35.71, 32.91, 31.50, 29.93, 29.81, 29.66, 29.63, 29.52, 29.51, 29.44,29.32, 29.30, 29.27, 27.24, 27.20, 27.17, 25.61, 24.46, 23.10, 22.54,14.04. Calc. mass for the C₄₇H₈₃NO₄: 725.6; found 726.3.

Compound 7011 (ALNY-237): Prepared by similar experimental conditionsused as for compound 7010, using alcohol 7009 (0.8 g, 1.27 mmol, 1.0eq), N,N-dimethylamino butyric acid hydrochloride (0.25 g, 1.53 mmol,1.2 eq), DIPEA (0.7 mL, 3.81 mmol, 3.0 eq), EDCI (0.26 g, 1.4 mmol, 1.1eq), DMAP (0.015 g, 0.12 mmol, 0.1 eq) in DCM gave 0.8 g (85%) of thepure product 7011. ¹H NMR (400 MHz, CDCl₃) δ 5.63-5.18 (m, 8H),4.85-4.46 (m, 1H), 2.84-2.70 (m, 4H), 2.36-2.25 (m, 6H), 2.23 (s, 6H),2.10-1.95 (m, 8H), 1.85-1.71 (m, 2H), 1.69-1.57 (m, 4H), 1.46 (d,J=21.4, 2H), 1.32 (ddd, J=21.9, 13.6, 7.7, 38H), 0.88 (t, J=6.8, 6H).¹³C NMR (101 MHz, cdcl₃) δ 173.11, 130.44, 130.41, 130.39, 130.34,128.19, 128.14, 128.12, 113.49, 78.53, 77.55, 77.23, 76.91, 74.06,58.96, 45.48, 38.57, 36.50, 35.93, 32.28, 31.75, 30.18, 30.06, 29.91,29.89, 29.77, 29.69, 29.57, 29.55, 29.52, 27.49, 27.45, 27.42, 25.86,24.71, 23.36, 22.96, 22.79, 14.29. Calc. mass for the C₄₈H₈₅NO₄: 739.6;found 740.3.

Example 88 Synthesis of Oxime- and Hydrazone Linked Lipids

Experimental Details

Compound 5006 (ALNY-175): To a flask containing EtOH (50 mL) was addeddimethylaminoethyl hydrazine dihydrochloride (1.00 g, 5.70 mmol) and theketone 5005 (2.00 g, 3.80 mmol). The mixture was heated at 60° C. for 16hours. After addition of Et₃N (0.5 mL), the reaction mixture wasevaporated. The residue was extracted with Et₂O and saturated NaHCO₃aq., and the organic layer was dried over MgSO₄, filtered andconcentrated. The crude was purified by silica gel column chromatography(CH₂Cl₂:MeOH:NH₃ aq.=95:5:0.5, R_(f)=0.29) to give compound 3 (1.78 g,2.91 mmol, 76%). Molecular weight for C₄₁H₇₈N₃ (M+H)⁺ Calc. 612.62;Found 612.5.

Compound 5007 (ALNY-187): 3-Dimethylamino-propionic acid hydrazide (RyanScientific, 500 mg, 3.89 mmol) in EtOH (10 mL) and the dilinoleyl ketone5005 (1.74 g, 3.31 mmol) in EtOH (20 mL) were mixed together. To thesolution was added acetic acid (0.038 mL, 0.662 mmol), and the reactionmixture was heated at 65° C. for 5 hours. After addition of Et₃N (0.5mL), the reaction mixture was evaporated. The residue was extracted withCH₂Cl₂ and saturated NaHCO₃ aq., and the organic layer was dried overMgSO₄, filtered and concentrated. The crude was purified by silica gelcolumn chromatography (CH₂Cl₂:MeOH:NH₃aq.=95:5:0.5, R_(f)=0.30) to givecompound 5007 (1.40 g, 2.19 mmol, 66%). Molecular weight for C₄₂H₇₈N₃O(M+H)⁺ Calc. 640.61; Found 640.5.

Compound 5008b: To a solution of 5008a (30 g, 56.8 mmol) in toluene, wasadded N-Hydroxyphthalimide (13.9 g, 85 mmol) and TPP (22.30 g, 85 mmol)under argon. The reaction mass was cooled to −5° C., to this was addedTEA (11.84 mL), followed by DEAD (13.14 ml). The reaction mass wasallowed to stir for 12 hrs at room temperature (TLC). It was thenfiltered through celite pad. The filtrate was evaporated at reducedpressure to obtain crude product, which was purified by silica gelchromatography to afford pure product, which was eluted at 3% diethylether and hexane to get the product 5008b (22.90 g, 60.50%) as paleyellow liquid ¹HNMR (400 MHz, CDCl₃,): δ 0.90 (6H, t, J=7.2 Hz), 1.2-1.4(34H, m), 1.66-1.70 (4H, m), 2.03-2.08 (8H, m), 2.78 (4H, t, J=12.8 Hz),4.22 (1H, m), 5.29-5.43 (8H, m), 7.74-7.76 (2H, m), 7.83-7.85 (2H, m).¹³CNMR (100 MHz, CDCl₃,): δ 14.3, 22.5, 24.9, 25.6, 27.2, 27.20, 29.3,29.3, 29.5, 29.5, 29.6, 29.7, 31.5, 32.4, 88.3, 123.3, 127.9, 129.0,130.1, 134.3, 164.3. MS: Molecular weight calculated for C₄₅H₇₁NO₃673.54; Found: 674.55 (M+H).

Compound 5010: ALY-SAN-031 (2.36 g, 3.50 mmol) was treated withhydrazine monohydrate (0.424 mL, 5.60 mmol) in CH₂Cl₂ (36 mL) and EtOH(4 mL) for 2 hours. After filtration of the resulting whiteprecipitation, the filtrate was concentrated. The residue was extractedwith Et₂O and saturated NaHCO₃ aq., and the organic layer was dried overMgSO₄, filtered and concentrated. The crude 5008 was used for next stepwithout further purification. R_(f)=0.44 (10% EtOAC in Hexane).Molecular weight for C₃₇H₇₀NO (M+H)⁺ Calc. 544.55; Found 544.2.

The compound 5008 was dissolved in EtOH (30 mL), and4-(dimethylamino)butan-2-one (Matrix Scientific, 500 mg, 4.34 mmol) andacetic acid (0.040 mL, 0.70 mmol) was added to the solution. Thereaction mixture was stirred at room temperature for 14 hours. Afteraddition of Et₃N (0.5 mL), the reaction mixture was evaporated. Theresidue was extracted with Et₂O and saturated NaHCO₃ aq., and theorganic layer was dried over MgSO₄, filtered and concentrated. The crudewas purified by silica gel column chromatography (Hexane:EtOAc=1:1) togive compound 5010 as a mixture of E/Z-isomers (1.90 g, 2.96 mmol, 85%,2 steps, R_(f)=0.39, 0.21 developed with Hexane:EtOAc=1:1). Molecularweight for C₄₃H₈₁N₂O (M+H)⁺ Calc. 641.63; Found 641.5.

Compound 5009: Compound 5006 (800 mg, 1.47 mmol) was dissolved in EtOH(15 mL), (Dimethylamino)acetone (Aldrich, 0.220 mL, 1.91 mmol) andacetic acid (0.017 mL, 0.294 mmol) were added to the solution then thereaction mixture was stirred at room temperature for 14 hours. Afteraddition of Et₃N (0.5 mL), the reaction mixture was evaporated. Theresidue was extracted with Et₂O and saturated NaHCO₃ aq., and theorganic layer was dried over MgSO₄, filtered and concentrated. The crudewas purified by silica gel column chromatography (Hexane:EtOAc=9:1) togive compound 5009 (868 mg, 1.38 mmol, 94%, R_(f)=0.22 developed withHexane:EtOAc=9:1). Molecular weight for C₄₂H₇₉N₂O (M+H)⁺ Calc. 627.62;Found 627.5.

Compound 5011: Compound 5006 (1.09 g, 2.00 mmol) was dissolved in EtOH(20 mL). 1-Methyl-4-piperidone (Aldrich, 0.320 mL, 2.60 mmol) and aceticacid (0.40 mL, 0.400 mmol) were added to the solution then the reactionmixture was stirred at room temperature for 14 hours. After addition ofEt₃N (0.5 mL), the reaction mixture was evaporated. The residue wasextracted with Et₂O and saturated NaHCO₃ aq., and the organic layer wasdried over MgSO₄, filtered and concentrated. The crude was purified bysilica gel column chromatography (CH₂Cl₂:MeOH:NH₄OH=97:3:0.3) to givecompound 5011 (1.11 g, 1.74 mmol, 87%, R_(f)=0.20 developed withCH₂Cl₂:MeOH:NH₄OH=97:3:0.3). Molecular weight for C₄₃H₇₉N₂O (M+H)⁺ Calc.639.62; Found 639.5.

Example 89 Synthesis of Other Lipids

Synthesis of 2051: To a solution of 2004 (50 g, 95 mmol) in DCM (400 ml)under Ar atmosphere, was added TEA (53 mL, 378 mmol) and DMAP (1.2 g,9.5 mmol) and stirred at room temperature under Ar atmosphere. Reactionmass was cooled to −5° C. and the solution of mesyl chloride (15 mL, 190mmol) in DCM (100 ml) was added slowly at temperature below −5° C. andallowed to warm to RT after addition. After 30 minutes (TLC), reactionmass was quenched with ice cold water (20 ml). Organic layer wasseparated, washed with 1N HCl (30 ml), water, brine, dried over sodiumsulfate and evaporated at reduced pressure to obtain pure product (55 g,95.5%) as yellow liquid. 1H NMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J=6.8),1.2-1.5 (m, 36H), 1.67 (m, 4H), 2.05 (q, 8H, J1=6.8, J2=6.8), 2.77 (t,4H, J=6.4), 2.99 (s, 3H), 4.71 (m, 1H) and 5.36 (m, 8H).

Synthesis of 2052: To a stirred solution of sodium cyanide (1.70 g,0.0330 mol) in DMF, was added compound 2051 (10 g, 0.0165 mol) in DMF(100 mL) slowly and heated to 55° C. for 24 hrs (TLC). It was thencooled to room temperature, diluted with water and extracted with ethylacetate several times. The combined organic layers were washed withwater, brine, dried over sodium sulfate and evaporated at reducedpressure to obtain crude product, which was purified silica gelchromatography using 1% ether/hexane to get the product as a pale yellowliquid (5.80 g, 62%). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (t, 6H, J=6.8 Hz),1.25 (m, 38H), 1.52 (m, 4H), 2.03 (q, 8H, J=6.8 Hz, J=6.8 Hz), 2.47 (m,1H), 2.76 (t, 4H, J=6.4 Hz), 5.32 (m, 8H).

Synthesis of 2053: To a cooled suspension of LAH (1.50 g, 0.0387 mol) inTHF (52 ml) at 0° C. under argon atmosphere, was added compound 2052(5.2 g, 0.0097 mol) in THF drop-wise. After addition, it was allowed towarm to RT and stirred for 20 hrs (TLC). It was cooled to 0° C. andquenched with saturated solution of sodium sulfate (10 ml) followed byethyl acetate. It was filtered through celite bed and washed with ethylacetate. The combined organic filtrate was evaporated at reducedpressure to obtain crude product, which was purified by silica gelchromatography using 10% ethyl acetate in hexane to get the product aspale brown liquid (3.70 g, 71%). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (t, 6H,J=6.8 Hz), 1.27 (m, 48H), 2.03 (q, 8H, 6.8 Hz, 6.8 Hz), 2.60 (d, 2H,J=4.0 Hz), 2.76 (t, 4H, J=6.4 Hz), 5.31 (m, 8H). ¹³C NMR (100 MHz,CDCl₃): δ 14.1, 22.6, 25.6, 26.8, 27.1, 27.2, 29.3, 29.5, 29.6, 30.1,31.5, 40.9, 45.2, 128.0, 130.1. Mass 543 (M+).

Synthesis of 2054: To a solution of compound 2053 (45 g, 0.083 mol) inDCM (450 mL) under argon atmosphere at 0° C., was added 2,6-Lutidine(19.3 mL, 0.166 mol) followed by benzyl chloroformate (12.1 mL, 0.0847mol) drop-wise. It was then warmed to 20° C. and stirred for one hour atthat temperature (TLC). Then it was diluted with DCM (200 ml), washedwith 10% citric acid (2×200 ml), water, brine and dried over anhydroussodium sulfate, evaporated at reduced pressure to obtain crude product,which was purified by silica gel chromatography using 3% ether/hexane toget the final product as pale brown liquid (36 g, 64%). ¹H NMR (400 MHz,CDCl₃): δ 0.87 (t, 6H, J=6 Hz), 1.28 (m, 44H), 2.02 (q, 8H, J₁=6.8 Hz,J₂=6.8 Hz), 2.76 (t, 4H, J=6.4 Hz), 3.11 (t, 2H, J=5.6 Hz), 4.67 (s,1H), 5.18 (s, 2H), 5.30 (m, 8H), 7.31 (m, 4H).

Synthesis of 2055: To a suspension of lithium aluminiumhydride (4.05 g,0.1066 mol) in THF (360 mL) under argon atmosphere at 0° C., was added asolution of 2054 (36 g, 0.0533 mol) in THF drop-wise. After addition, itwas allowed to warm to room temperature and stirred for 15 hours (TLC).The reaction mass was cooled to 0° C. and quenched with saturatedsolution of sodium sulfate followed by ethyl acetate. It was filteredthrough celite bed and washed with ethyl acetate. Combined filtrateswere evaporated and purified by silica gel using 100% methanol to getthe final product 26 g, 87%). ¹H NMR (400 MHz, CDCl₃): δ 0.87 (t, 6H,J=6.8 Hz), 1.27 (m, 42H), 2.03 (q, 8H, J₁=6.8 Hz, J₂=6.8 Hz), 2.45 (s,3H), 2.49 (d, 2H, J=6 Hz), 2.76 (t, 4H, J=6.4 Hz), 5.30 (m, 8H).

Synthesis of 2055a: Compound 2055 (4 g, 0.0072 mol) was dissolved in DCM(40 mL) under argon atmosphere and cooled to 0° C. To this solution2,6-Lutidine (1.7 mL, 0.0144 mol) was added drop-wise followed by benzylchloroformate (1.0 mL, 0.0074 mol). It was then allowed to warm to 20°C. and stirred for one hour (TLC). Then it was diluted with DCM (200ml), washed with 10% citric acid (2×200 mL), water and brine. Theorganic layer was dried over anhydrous sodium sulfate and evaporated atreduced pressure to obtain crude product, which was purified by silicagel using 3% ether/hexane to get the final product (3.80 g, 76%). ¹H NMR(400 MHz, CDCl₃): δ 0.87 (t, 6H, J=6.8 Hz), 1.20 (m, 44H), 2.02 (q, 8H,J₁=6.8 Hz, J₂=6.8 Hz), 2.76 (t, 4H, J=6.4 Hz), 2.89 (d, 3H, J=6 Hz),3.14 (m, 2H), 5.12 (s, 2H), 5.30 (m, 8H), 7.26 (m, 4H).

Synthesis of 2056: To a suspension of lithium aluminiumhydride (0.52 g,0.0138 mol) in THF under argon atmosphere at 0° C., was added a solutionof 2055a (3.80 g, 0.0055 mol) in THF (38 mL) dropwise. After addition,it was allowed to warm to room temperature and stirred for 15 hours(TLC). The reaction mass was cooled to 0° C. and quenched with saturatedsolution of sodium sulfate followed by ethyl acetate. Whole mass wasfiltered through celite bed and washed with ethyl acetate. Combinedfiltrates were evaporated at reduced pressure to obtain crude product,which was purified silica gel chromatography using 100% methanol to getthe final product as colorless liquid (2.20 g, 70%) 1H NMR (400 MHz,CDCl₃): δ 0.87 (t, 6H, J=6.8 Hz), 1.21 (m, 44H), 2.03 (q, 8H, J=6.8 Hz,J=6.4 Hz), 2.18 (s, 6H), 2.76 (t, 4H, J=6.4 Hz), 5.30 (m, 8H). ¹³C NMR(100 MHz, CDCl₃): δ 14.0, 22.4, 25.5, 26.5, 27.0, 27.1, 29.2, 29.4,29.6, 30.0, 31.4, 32.1, 35.6, 45.9, 64.8, 127.8, 130.0. ELSD: 99.0%Mass: 570.2 (M⁺)

Synthesis of 2062 (ALNY-141)

Synthesis of 2061: To a solution of 2055 (5 g, 0.0089 mol) in 100 ml ofDCM under argon at 0° C. was added NaBH(OAc)₃ (2.30 g, 0.0106 mol)Stirred for 20 minutes. Aldehyde (1.70 g, 0.0082 mol) in 700 ml of DCMwas added slowly to the reaction mass over a period of 45 minutes. Afteraddition the reaction mass was allowed to stir at RT for 15-20 minutes.TLC showed the absence of starting material. The reaction mass waswashed with sat. NaHCO₃ (2×500 ml) and water (500 ml). The aqueous layerwas re-extracted with DCM (500 ml). The combined organic layer waswashed with brine (500 ml). The organic layer was dried over Na₂SO₄,filtered and concentrated. The crude obtained was purified by silica gelchromatography and Hexane/Diethyl ether as eluent. The product goteluted at 8% of ether in hexane as brown liquid (yield, 6.40 g, 96%). ¹HNMR: (400 MHz, CDCl₃):_(—)0.89 (t, 6H, J=7.2 Hz), 1.26-1.43 (m, 40H),1.85 (m, 1H), 2.06 (q, 8H, J1=6.8 Hz, J2=6.8 Hz), 2.15 (s, 2H), 2.20 (s,3H), 2.45 (m, 2H), 2.77 (t, 4H, J=6 Hz), 2.95 (s, 3H), 3.35 (m, 2H),5.12 (s, 2H), 5.32 (m, 8H), 7.35 (m, 5H).

Synthesis of 2062: To a suspension of lithium aluminiumhydride (0.751 g,0.0198 mol) in THF under argon atmosphere at 0° C., was added a solutionof 2061 (5.7 g, 0.0076 mol) in THF drop-wise. After addition, it wasallowed to warm to room temperature and stirred for 15 hours (TLC). Thereaction mass was cooled to 0° C. and quenched with saturated solutionof sodium sulfate (50 ml) followed by ethyl acetate (100 ml). It wasfiltered through celite bed and washed with ethyl acetate. Combinedfiltrates were evaporated at reduced pressure to obtain crude product,which was purified silica gel chromatography usingDCM/Ethylacetate/Chloroform/Methanol as eluent. The product eluted at 3%chloroform in methanol as brown liquid (3.80 g, 80%) ¹H NMR: (400 MHz,CDCl₃): 0.89 (t, 6H, J=6.8 Hz), 1.26-1.37 (m, 40H), 1.42 (m, 1H), 2.06(q, 8H, J1=6.8 Hz, J2=6.8 Hz), 2.15 (d, 2H, J=7.2 Hz), 2.20 (s, 3H),2.29 (s, 6H), 2.45 (s, 4H), 2.78 (t, 4H, J=6.4 Hz), 5.36 (m, 8H). ¹³CNMR: (100 MHz, CDCl₃): 14.1, 22.6, 25.6, 26.6, 27.2, 27.22, 28.9, 29.3,29.6, 29.7, 30.1, 31.5, 32.2, 35.8, 43.2, 45.7, 56.2, 57.2, 63.3, 127.9,130.2. HPLC ELSD: 100% Mass: 627.53

Example 90 Synthesis of Reverse Ketals

Synthesis of 2001: To 500 ml of ethanol cooled below 0° C. usingice-salt mixture was added 10 ml of Conc. H₂SO₄ slowly. Linoleic acid(100 g, 357 mmol) in 500 ml of ethanol was added to the above solutionslowly by maintaining the temperature below 0° C. After addition thereaction mass was warmed to RT and then refluxed for 5 hrs (TLC). It wasthen cooled to room temperature and neutralized by sat. NaHCO₃ solution.The resulting solution was concentrated to remove excess of solvent. Theresidue was diluted with water (1000 ml) and extracted with DCM (6×500ml). The combined organic layer was washed with brine (1000 ml), anddried over sodium sulfate, filtered and evaporated at reduced pressureto obtain pure product (109.80 g, 99%) as a pale yellow liquid, whichwas taken as such for the next stage. ¹H NMR (CDCl₃): δ 0.89 (t, 3H,J=6.8 Hz), 1.24-1.31 (m, 17H), 1.62 (m, 2H, J=10 Hz), 2.04 (q, 4H,J₁=6.8 Hz, J₂=6.8 Hz), 2.29 (t, 2H, J=7.6 Hz), 2.76 (t, 2H, J=6.4 Hz),4.13 (q, 2H, J₁=7.2 Hz, J₂=7.2 Hz), 5.34 (m, 4H). ¹³C NMR (CDCl₃): δ13.9, 14.1, 22.5, 24.9, 25.5, 27.1, 29.0, 29.1, 29.3, 29.5, 31.4, 34.2,60.0, 127.8, 127.9, 129.9, 130.0, 173.6.

Synthesis of 2002: To 660 ml of freshly distilled toluene in a 2 Lmultineck RB flask fitted with reflux condenser under argon was addedsodium pieces (41.1 g, 1.785 mol). To this was added TMSCl (192 mL,1.499 mol) slowly and heated to 40° C. after addition. Then a solutionof 2001 (110 g, 0.357 mol) in 275 ml of freshly distilled toluene wasadded slowly by maintaining the reaction temperature at 40° C. over aperiod of 1 hr. It was then refluxed for 2-3 hrs. After 3 hrs thereaction mass turned pale purple in color (TLC). Heating was stopped andthe reaction mass was cooled to room temperature, filtered through a padof celite and washed with toluene. (CAUTION: The reaction mixturecontained unreacted sodium pieces). The filtrate obtained was stirredwith 3 L of sat. NH₄Cl solution for 15-20 minutes until the silyl etherconverted to the required -keto alcohol. The organic layer was separatedand the aqueous layer was washed with ethyl acetate (3×1000 ml). Thecombined organic layer was washed with brine (1 L), dried over sodiumsulfate, filtered and evaporated at reduced pressure to obtain the crudematerial, which was purified by silica gel chromatography usinghexane/ethyl acetate as eluent. The product got eluted at 3% ethylacetate in hexane to get 2002 (44 g, 47%) as a pale yellow liquid. ¹HNMR (400 MHz, CDCl₃): δ 0.89 (t, 6H, J=7.2 Hz), 1.2-1.3 (m, 30H), 1.53(m, 1H), 1.62 (m, 2H), 1.8 (m, 1H), 2.04 (q, 8H, J₁=6.8 Hz, J₂=6.8 Hz),2.43 (m, 2H), 2.76 (t, 4H, J=6.4 Hz), 3.49 (d, 1H, J=4.8 Hz) 4.16 (m,1H), 5.34 (m, 8H). ¹³C NMR (100 MHz, CDCl₃): δ 14.1, 22.6, 23.6, 24.8,25.6, 27.2, 29.1, 29.2, 29.24, 29.3, 29.4, 29.56, 29.6, 31.5, 33.7,37.8, 76.3, 127.8, 127.9, 129.9, 130.0, 212.4.

Synthesis of 2003: A solution of 2002 (44 g, 83 mol) in methanol/DCMmixture (490 mL, DCM was added to make the solution homogeneous) underargon was cooled below 0° C. using ice-salt mixture. Sodium borohydride(4.7 g, 125 mmol) was added in one lot to the reaction mass. Thesuspension was stirred for 2 hrs, and the mass temperature slowly raisedto RT. After 2 hrs the reaction mass became homogeneous. TLC showed theabsence of starting material. The reaction was quenched with 100 ml ofwater, and concentrated to remove excess solvent. The residue obtainedwas again diluted with water (500 ml), and extracted with DCM (4×500ml). The combined organic layer was washed with brine (500 ml), driedover sodium sulfate, filtered and evaporated at reduced pressure toobtain the crude product, which was purified by silica gelchromatography using hexane/ethyl acetate as eluent. The product elutedfrom 4% to 20% of ethyl acetate in hexane to get 2003 (36 g, 82%) as awhite semisolid. ¹H NMR (CDCl₃): δ 0.89 (t, 6H, J=6.8 Hz), 1.2-1.5 (m,36H), 1.78 (d, 1H, J=4 Hz), 1.95 (d, 1H, J=4 Hz), 2.04 (q, 8H, J₁=6.8Hz, J₂=6.8 Hz), 2.77 (t, 4H, J=6.4 Hz), 3.40 (m, 1H), 3.61 (m, 1H), 5.34(m, 8H). ¹³C NMR (CDCl₃): δ13.7, 22.2, 25.2, 25.3, 25.7, 26.8, 28.4,28.9, 29.0, 29.1, 29.3, 30.8, 31.1, 33.2, 74.1, 74.3, 127.5, 127.6,129.7, 129.8. MS: Molecular weight calculated for C₃₆H₆₆O₂ 530.51;Found: 531.52 (M+H)

Example 91 Synthesis of ALNY-152

To a solution of diol 2003 (2.1 g, 4.0 mmol), ketone 2004 (0.50 g, 4.3mmol) in toluene was added the PTSA (0.86 g, 5 mmol) and refluxed underDean-Stock apparatus until there is no starting material left. Cooledthe reaction mixture, evaporated, directly loaded on columnchromatography and purified using 0-10% MeOH in CH₂Cl₂ to get 1.25 g ofthe pure compound ALNY-152 in 50% yields. ¹H NMR (400 MHz, CDCl₃) δ5.46-5.24 (m, 8H), 4.09-3.93 (m, 1H), 3.65-3.42 (m, 2H), 2.84-2.68 (m,5H), 2.47-2.30 (m, 3H), 2.27-2.16 (m, 7H), 2.04 (q, J=6.8, 9H), 1.82(qd, J=12.4, 7.8, 2H), 1.46 (t, J=19.0, 7H), 1.40 (s, 38H), 0.99-0.76(m, 6H). Calc. mass for the C₄₂H₇₇NO₂: 628.07; found 628.5.

Example 92 Synthesis of ALNY-220

Using a similar procedure used for the synthesis of ALNY-152, using 1.06g (2 mmol) of 2003 and 400 mg (2.5 mmol) of the amine 2006, 0.7 g (56%)the ketalized product ALNY-220 was isolated after column purification.¹H NMR (400 MHz, CDCl₃) δ 5.44-5.24 (m, 8H), 5.02-4.83 (m, 1H), 3.87(dd, J=17.2, 14.0, 1H), 3.51 (dd, J=15.2, 8.5, 1H), 2.72 (dt, J=9.5,5.0, 4H), 2.32-2.25 (m, 2H), 2.22 (s, 6H), 2.11-1.94 (m, 9H), 1.68-1.38(m, 11H), 1.38-1.18 (m, 35H), 0.84 (dt, J=10.7, 5.9, 6H). Calc. mass forthe C₄₂H₇₇NO₂: 628.07; found 628.5.

Preparation of Compound ALNY-158

Preparation of Compound 2008: Using similar ketalyzation procedure ascompound ALNY-220, using N-Cbz-4-amino cyclohexanone 2007 (1.5 g, 6.0mmol, 1.0 eq), dilinoleyl diol 2003 (3.38 g, 6.0 mmol, 1.0 eq) and PTSA(0.11 g, 0.6 mmol, 0.1 eq), which gave 3.31 g (69%) of the purecorresponding ketal 2008. ¹H NMR (400 MHz, CDCl₃) δ 7.45-7.24 (m, 5H),5.47-5.24 (m, 8H), 5.06 (d, J=10.8, 1H), 4.63 (d, J=7.4, 1H), 3.97 (s,1H), 3.55 (d, J=3.7, 2H), 2.76 (t, J=6.4, 4H), 2.04 (q, J=6.7, 8H), 1.91(s, 2H), 1.81-1.59 (m, 3H), 1.56-1.42 (m, 8H), 1.42-1.15 (m, 31H), 0.88(t, J=6.8, 6H). Calc. mass for the C₅₀H₈₁NO₄: 759.62; found 786 (+Na).

Preparation of Compound 2009: Used similar experimental proceduredescribed earlier, using N-Cbz ketal 24 (2.42 g, 3.0 mmol, 1.0 eq), 1MLAH in THF (4.6 mL, 4.6 mmol, 1.5 eq), this gave 2.17 g of the compound2009 in quantitative yields. ¹H NMR (400 MHz, CDCl₃) δ 5.49-5.12 (m,8H), 4.69 (s, 1H), 3.98 (d, J=5.9, 1H), 3.56 (d, J=3.7, 1H), 2.76 (t,J=6.2, 4H), 2.37 (s, 3H), 2.13-1.93 (m, 8H), 1.92-1.63 (m, 5H),1.60-1.40 (m, 8H), 1.40-1.14 (m, 34H), 0.88 (t, J=6.8, 6H). Calc. massfor the C₄₃H₇₇NO₂: 639.6; found 640.3.

Preparation of Compound 2010: To a stirred solution of N-methyl ketal2009 (2.0 g, 3.0 mmol, 1.0 eq), triethyl amine (1.3 mL, 9.0 mmol, 3.0eq) in dichloromethane at 0° C. was added solid Z-OSu portions wise andcontinued the reaction at room temperature under argon for overnight.After completion of the reaction, diluted with dichloromethane, washedwith water, brine, dried on MgSO₄, concentrated and purified by columnchromatography using hexane: ethylacetate (20%) as gradients to get 1.94g (80%) of the pure Cbz-protected ketal 2009. ¹H NMR (400 MHz, CDCl₃) δ7.50-7.16 (m, 5H), 5.49-5.24 (m, 8H), 5.15 (d, J=15.4, 2H), 4.04 (dd,J=59.3, 9.4, 1H), 3.72-3.43 (m, 1H), 2.77 (dd, J=13.2, 6.5, 8H), 2.04(q, J=6.7, 8H), 1.93-1.68 (m, 4H), 1.66-1.55 (m, 3H), 1.48 (d, J=5.1,6H), 1.38-1.17 (m, 30H), 0.88 (t, J=6.7, 6H). Calc. mass for theC₅₁H₈₃NO₄: 773.6; found 774.3.

Preparation of Compound ALNY-158: Used similar experimental procedure asdescribed earlier, using N-Cbz-ketal 2010 (1.48 g, 1.85 mmol, 1.0 eq),1M LAH in THF (2.77 mL, 2.77 mmol, 1.5 eq), this gave 1.0 g of thecompound ALNY-158 in 79% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.48-5.13 (m,8H), 3.97 (d, J=5.8, 1H), 3.55 (d, J=4.2, 1H), 2.76 (t, J=6.3, 4H), 2.27(s, 6H), 2.23-2.12 (m, 1H), 2.04 (q, J=6.7, 8H), 1.80 (dt, J=16.5, 10.8,3H), 1.65 (t, J=10.2, 1H), 1.60-1.42 (m, 8H), 1.42-1.16 (m, 32H), 0.88(t, J=6.8, 6H). ¹³C NMR (101 MHz, cdcl₃) δ 130.41, 130.32, 128.20,128.13, 107.86, 107.39, 81.06, 80.95, 78.03, 77.98, 77.54, 77.22, 76.90,62.74, 62.69, 42.07, 42.02, 37.20, 35.80, 35.18, 34.08, 33.59, 33.43,31.74, 30.12, 29.98, 29.91, 29.87, 29.78, 29.69, 29.64, 29.57, 29.50,29.47, 27.45, 27.42, 26.55, 26.43, 26.41, 26.37, 26.13, 25.85, 25.80,22.79, 14.29. Calc. mass for the C₄₄H₇₉NO₂: 653.6; found 654.3.

Preparation of ALNY-155

Preparation of Compound 2012: Used similar ketalyzation procedure ascompound ALNY-152, using 1-N-Cbz-pyrrolidinone 2011 (1.61 g, 7.37 mmol,1.0 eq), dilinoleyl diol 2003 (4.16 g, 7.37 mmol, 1.0 eq) and PTSA (0.14g, 0.73 mmol, 0.1 eq), which gave 3.2 g (57%) of the pure correspondingketal 2012. ¹H NMR (400 MHz, CDCl₃) δ 7.41-7.26 (m, 5H), 5.48-5.23 (m,8H), 5.12 (s, 2H), 3.98 (m, 1H), 3.66-3.29 (m, 5H), 2.76 (t, J=6.4, 4H),2.00 (dq, J=14.5, 6.9, 11H), 1.54-1.15 (m, 41H), 0.88 (t, J=6.8, 6H).Calc. mass for the C₄₈H₇₇NO₄: 731.5; found 732.0.

Preparation of ALNY-155: Used similar experimental procedure asdescribed earlier, using N-Cbz-ketal 2012 (1.01 g, 1.33 mmol, 1.0 eq),1M LAH in THF (2.0 mL, 2.0 mmol, 1.5 eq), this gave 0.61 g of thecompound ALNY-155 in 72% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.47-5.16 (m,8H), 4.00-3.77 (m, 1H), 2.76 (t, J=6.4, 4H), 2.62-2.37 (m, 3H), 2.30 (s,3H), 2.15-1.90 (m, 11H), 1.57-1.15 (m, 42H), 0.88 (t, J=6.8, 6H). ¹³CNMR (101 MHz, cdcl₃) δ 130.41, 130.32, 128.21, 128.13, 115.42, 114.88,114.78, 81.46, 81.22, 78.51, 78.21, 77.54, 77.22, 76.91, 66.74, 66.55,66.34, 55.28, 55.00, 54.84, 42.70, 39.13, 38.78, 33.38, 33.20, 31.74,29.88, 29.66, 29.64, 29.57, 29.51, 29.47, 29.41, 27.45, 27.42, 26.41,26.25, 26.22, 25.85, 22.79, 14.29. Calc. mass for the C41H73NO2: 611.5;found 612.3.

Preparation of ALNY-156

Preparation of Compound 2014: Used similar ketalyzation procedure ascompound ALNY-152, using 1-N-Cbz-piperidone 2013 (1.0 g, 4.29 mmol, 1.0eq), dilinoleyl diol 2003 (2.4 g, 4.29 mmol, 1.0 eq) and PTSA (0.08 g,0.43 mmol, 0.1 eq), which gave 2.74 g (71%) of the pure correspondingketal 2014. ¹H NMR (400 MHz, CDCl₃) δ 7.42-7.21 (m, 5H), 5.46-5.22 (m,8H), 5.11 (s, 2H), 4.01 (d, J=7.1, 1H), 3.68-3.43 (m, 5H), 2.81-2.66 (m,4H), 2.04 (q, J=6.7, 8H), 1.76-1.40 (m, 9H), 1.40-1.15 (m, 32H), 0.88(t, J=6.8, 6H). Calc. mass for the C₄₉H₇₉NO₄: 745.6; found 746.3.

Preparation of ALNY-156: Used similar experimental procedure asdescribed earlier, using N-Cbz-ketal 2014 (2.72 g, 3.52 mmol, 1.0 eq),1M LAH in THF (5.27 mL, 5.27 mmol, 1.5 eq), this gave 2.13 g of thecompound ALNY-156 in 93% yield. ¹H NMR (400 MHz, CDCl₃) δ 5.52-5.20 (m,8H), 4.09-3.92 (m, 1H), 3.61 (s, 1H), 2.79 (t, J=6.5, 4H), 2.49 (s, 4H),2.31 (s, 3H), 2.07 (q, J=6.8, 8H), 1.75 (dd, J=11.5, 5.8, 4H), 1.52 (m,5H), 1.43-1.19 (m, 31H), 0.91 (t, J=6.9, 6H). ¹³C NMR (101 MHz, CDCl₃) δ130.22, 130.12, 128.01, 127.92, 105.99, 105.51, 80.73, 77.82, 77.35,77.23, 77.03, 76.71, 53.80, 53.61, 53.54, 45.98, 38.07, 36.53, 35.24,33.27, 31.54, 29.74, 29.70, 29.66, 29.46, 29.45, 29.36, 29.27, 27.24,27.21, 26.24, 26.14, 25.64, 22.59, 14.10. Calc. mass for the C₄₂H₇₅NO₂:625.5; found 626.2.

Preparation of ALNY-227

To a stirred solution of dilinoleyl diol 2003 (1.0 g, 1.8 mmol, 1.0 eq),N,N-dimethyl butyric acid hydrochloride 2015 (0.89 g, 5.37 mmol, 3.0eq), DIPEA (2.0 mL, 10.8 mmol, 6.0 eq) in dichloromethane was addedsolid EDCI (0.76 g, 3.96 mmol, 2.2 eq), DMAP (0.04 g, 0.36 mmol, 0.2 eq)portions wise and continued at room temperature. After completion of thereaction was diluted with dichloromethane, washed with saturatedsolution of NaHCO₃, brine and combined organics were dried over MgSO₄,evaporated the solvent, and the reaction mixture is purified by columnchromatography using dichloromethane and methanol (5%) as gradients toget pure bis-ester ALNY-227.

Preparation of bis-MC2 derivative ALNY-239

Prepared the bis-MC2 analog (ALNY-239) by similar experimentalconditions as compound ALNY-238, using dilinoleyl diol 2003 (1.0 g, 1.8mmol, 1.0 eq), N,N-dimethyl propionic acid hydrochloride (0.82 g, 5.37mmol, 3.0 eq), DIPEA (2.0 mL, 10.8 mmol, 6.0 eq) in dichloromethane wasadded solid EDCI (0.76 g, 3.96 mmol, 2.2 eq), DMAP (0.04 g, 0.36 mmol,0.2 eq) to get pure bis ester of the MC2 derivative ALNY-239. Calc. massfor the C₄₆H₈₄N₂O₄: 729.1; found 729.5.

The various embodiments described above can be combined to providefurther embodiments. All of the U.S. patents, U.S. patent applicationpublications, foreign patents, foreign patent applications andnon-patent publications referred to in this specification and/or listedin the Application Data Sheet, U.S. Ser. No. 61/113,179, filed Nov. 10,2008; U.S. Ser. No. 61/154,350, filed Feb. 20, 2009; U.S. Ser. No.61/171,439, filed Apr. 21, 2009; U.S. Ser. No. 61/185,438, filed Jun. 9,2009; U.S. Ser. No. 61/225,898, filed Jul. 15, 2009; and U.S. Ser. No.61/234,098, filed Aug. 14, 2009 are incorporated herein by reference, intheir entirety. Aspects of the embodiments can be modified, if necessaryto employ concepts of the various patents, applications and publicationsto provide yet further embodiments.

These and other changes can be made to the embodiments in light of theabove-detailed description. In general, in the following claims, theterms used should not be construed to limit the claims to the specificembodiments disclosed in the specification and the claims, but should beconstrued to include all possible embodiments along with the full scopeof equivalents to which such claims are entitled. Accordingly, theclaims are not limited by the disclosure.

1. A lipid of formula I or a salt or isomer thereof, wherein R₁ and R₂are each independently for each occurrence optionally substitutedC₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀ alkoxy, optionallysubstituted C₁₀-C₃₀ alkenyl, optionally substituted C₁₀-C₃₀ alkenyloxy,optionally substituted C₁₀-C₃₀ alkynyl, optionally substituted C₁₀-C₃₀alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl, or -linker-ligand;R₃ is independently for each occurrence H, optionally substituted C₁-C₁₀alkyl, optionally substituted C₂-C₁₀ alkenyl, optionally substitutedC₂-C₁₀ alkynyl, optionally substituted alkylheterocycle, optionallysubstituted heterocycloalkyl, optionally substituted alkylphosphate,optionally substituted phosphoalkyl, optionally substitutedalkylphosphorothioate, optionally substituted phosphorothioalkyl,optionally substituted alkylphosphorodithioate, optionally substitutedphosphorodithioalkyl, optionally substituted alkylphosphonate,optionally substituted phosphonoalkyl, optionally substituted amino,optionally substituted alkylamino, optionally substituteddi(alkyl)amino, optionally substituted aminoalkyl, optionallysubstituted alkylaminoalkyl, optionally substituted di(alkyl)aminoalkyl,optionally substituted hydroxyalkyl, optionally substituted polyethyleneglycol (PEG, mw 100-40K), optionally substituted mPEG (mw 120-40K),optionally substituted heteroaryl, or optionally substitutedheterocycle, or linker-ligand; X and Y are each independently —O—, —S—,alkylene, —N(Q)-, —C(O)—, —O(CO), —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —OS(O)(Q₂)O—, or —OP(O)(Q₂)O—; Q is H, alkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl; Q₁ isindependently for each occurrence O or S; Q₂ is independently for eachoccurrence O, S, N(Q)(Q), alkyl or alkoxy; A₁ and A₂ are eachindependently —O—, —S—, —CH₂—, —CHR⁵—, —CR⁵R⁵—, —CHF— or —CF₂—; eachoccurrence of R⁵ is independently H, halo, cyano, hydroxy, amino,optionally substituted alkyl, optionally substituted alkoxy, oroptionally substituted cycloalkyl; Z is N, or C(R₃); and m and n areeach independently 0 to 5, where m and n taken together result in a 3,4, 5, 6, 7 or 8 member ring.
 2. The lipid of claim 1, selected from

and salts thereof.
 3. A lipid of formula XXXV or a salt or isomerthereof, wherein R₁ and R₂ are each independently for each occurrenceoptionally substituted C₁₀-C₃₀ alkyl, optionally substituted C₁₀-C₃₀alkoxy, optionally substituted C₁₀-C₃₀ alkenyl, optionally substitutedC₁₀-C₃₀ alkenyloxy, optionally substituted C₁₀-C₃₀ alkynyl, optionallysubstituted C₁₀-C₃₀ alkynyloxy, or optionally substituted C₁₀-C₃₀ acyl,or -linker-ligand; R₃ is independently for each occurrence H, optionallysubstituted C₁-C₁₀ alkyl, optionally substituted C₂-C₁₀ alkenyl,optionally substituted C₂-C₁₀ alkynyl, optionally substitutedalkylheterocycle, optionally substituted heterocycloalkyl, optionallysubstituted alkylphosphate, optionally substituted phosphoalkyl,optionally substituted alkylphosphorothioate, optionally substitutedphosphorothioalkyl, optionally substituted alkylphosphorodithioate,optionally substituted phosphorodithioalkyl, optionally substitutedalkylphosphonate, optionally substituted phosphonoalkyl, optionallysubstituted amino, optionally substituted alkylamino, optionallysubstituted di(alkyl)amino, optionally substituted aminoalkyl,optionally substituted alkylaminoalkyl, optionally substituteddi(alkyl)aminoalkyl, optionally substituted hydroxyalkyl, optionallysubstituted polyethylene glycol (PEG, mw 100-40K), optionallysubstituted mPEG (mw 120-40K), optionally substituted heteroaryl, oroptionally substituted heterocycle, or linker-ligand; or, each R₃ takentogether with the atom to which they are attached are a 3-8 memberedoptionally substituted cycloalkyl group or a 3-8 membered optionallysubstituted heterocycle group; X and Y are each independently —O—, —S—,alkylene, —N(Q)-, —C(O)—, —O(CO)—, —OC(O)N(Q)-, —N(Q)C(O)O—, —C(O)O,—OC(O)O—, —OS(O)(Q₂)O—, or —OP(O)(Q₂)O—; Q is H, alkyl, ω-aminoalkyl,ω-(substituted)aminoalkyl, ω-phosphoalkyl, or ω-thiophosphoalkyl; Q₁ isindependently for each occurrence O or S; and, Q₂ is independently foreach occurrence O, S, N(Q)(Q), alkyl or alkoxy;
 4. The lipid of claim 3,selected from

and salts thereof.
 5. A lipid particle comprising a lipid of claim
 1. 6.(canceled)
 7. The lipid particle of claim 5, wherein the particlefurther comprises a neutral lipid and a lipid capable of reducingaggregation.
 8. The lipid particle of claim 5, wherein the lipidparticle consists essentially of: a lipid of claim 1; a neutral lipidselected from DSPC, DPPC, POPC, DOPE and SM; sterol; and PEG-DMG, in amolar ratio of about 20-60% lipid:5-25% neutral lipid:25-55%sterol:0.5-15% PEG-DMG or PEG-DMA.
 9. The lipid particle of claim 5,further comprising a therapeutic agent.
 10. The lipid particle of claim9, wherein the therapeutic agent is a nucleic acid.
 11. The lipidparticle of claim 10, wherein the nucleic acid is a plasmid.
 12. Thelipid particle of claim 10, wherein the nucleic acid is animmunostimulatory oligonucleotide.
 13. The lipid particle of claim 10,wherein the nucleic acid is selected from an siRNA, an antisenseoligonucleotide, a microRNA, an antagomir, an aptamer, and a ribozyme.14. The lipid particle of claim 13, wherein the nucleic acid is ansiRNA.
 15. A pharmaceutical composition comprising a lipid particle ofclaim 10 and a pharmaceutically acceptable excipient, carrier, ordiluent.
 16. A method of modulating the expression of a target gene in acell, comprising providing to a cell the lipid particle of claim
 9. 17.The method of claim 16, wherein the therapeutic agent is selected froman siRNA, an antagomir, an antisense oligonucleotide, and a plasmidcapable of expressing an siRNA, a ribozyme, an aptamer or an antisenseoligonucleotide.
 18. The method of claim 16, wherein the nucleic acid isa plasmid that encodes the polypeptide or a functional variant orfragment thereof, such that expression of the polypeptide or thefunctional variant or fragment thereof is increased.
 19. A method oftreating a disease or disorder characterized by overexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of claim 15, wherein the therapeutic agent isselected from an siRNA, a microRNA, an antisense oligonucleotide, and aplasmid capable of expressing an siRNA, a microRNA, or an antisenseoligonucleotide, and wherein the siRNA, microRNA, or antisense RNAcomprises a polynucleotide that specifically binds to a polynucleotidethat encodes the polypeptide, or a complement thereof.
 20. A method oftreating a disease or disorder characterized by underexpression of apolypeptide in a subject, comprising providing to the subject thepharmaceutical composition of claim 15, wherein the therapeutic agent isa plasmid that encodes the polypeptide or a functional variant orfragment thereof.
 21. A method of inducing an immune response in asubject, comprising providing to the subject the pharmaceuticalcomposition of claim 15, wherein the therapeutic agent is animmunostimulatory oligonucleotide.
 22. The method of claim 21, whereinthe pharmaceutical composition is provided to the patient in combinationwith a vaccine or antigen.
 23. A vaccine comprising the lipid particleof claim 11 and an antigen associated with a disease or pathogen. 24.The vaccine of claim 23, wherein said antigen is a tumor antigen. 25.The vaccine of claim 23, wherein said antigen is a viral antigen, abacterial antigen, or a parasitic antigen.
 26. The lipid particle ofclaim 9, wherein the molar ratio is 52% lipid:5% neutral lipid:30%sterol:13% PEG-DMG.
 27. The method of claim 16, wherein the target geneis selected from Factor VII, Eg5, PCSK9, TPX2, apoB, SAA, TTR, RSV, PDGFbeta gene, Erb-B gene, Src gene, CRK gene, GRB2 gene, RAS gene, MEKKgene, JNK gene, RAF gene, Erk1/2 gene, PCNA(p21) gene, MYB gene, JUNgene, FOS gene, BCL-2 gene, Cyclin D gene, VEGF gene, EGFR gene, CyclinA gene, Cyclin E gene, WNT-1 gene, beta-catenin gene, c-MET gene, PKCgene, NFKB gene, STAT3 gene, survivin gene, Her2/Neu gene, SORT1 gene,XBP1 gene, topoisomerase I gene, topoisomerase II alpha gene, p73 gene,p21 (WAF1/CIP1) gene, p27 (KIP1) gene, PPM1D gene, RAS gene, caveolin Igene, MIB I gene, MTAI gene, M68 gene, tumor suppressor genes, and p53tumor suppressor gene.
 28. The method of claim 27, wherein the targetgene contains one or more mutations.
 29. A lipid particle comprising alipid of claim
 3. 30. The lipid particle of claim 29, wherein theparticle further comprises a neutral lipid and a lipid capable ofreducing aggregation.
 31. The lipid particle of claim 29, furthercomprising a therapeutic nucleic acid.
 32. The lipid particle of claim29, wherein the nucleic acid is an siRNA.
 33. A pharmaceuticalcomposition comprising a lipid particle of claim 32 and apharmaceutically acceptable excipient, carrier, or diluent.
 34. A methodof modulating the expression of a target gene in a cell, comprisingproviding to a cell the lipid particle of claim 32.